Sulfated oligosaccharide derivatives

ABSTRACT

The invention relates to compounds which are polysulfated oligosaccharide derivatives having activity as inhibitors of heparan sulfate-binding proteins and inhibitors of the enzyme heparanase; methods for the preparation of the compounds; compositions comprising the compounds, and use of the compounds and compositions thereof for the antiangiogenic, antimetastatic, anti-inflammatory, antimicrobial, anticoagulant and/or antithrombotic treatment, lowering of blood triglyceride levels and inhibition of cardiovascular disease of a mammalian subject.

TECHNICAL FIELD

The invention described herein relates to compounds having activity asinhibitors of heparan sulfate-binding proteins and as inhibitors of theenzyme heparanase. In particular, the invention is directed to sulfatedoligosaccharide derivatives, although the scope of the invention is notnecessarily limited thereto. Specifically, the invention relates topolysulfated oligosaccharide derivatives, the derivatisation beingpreferably at C-1 of the reducing end and/or C-6 of the non-reducing endmonosaccharide unit. The invention also relates to methods for thepreparation of the compounds, compositions comprising the compounds, anduse of the compounds and compositions thereof for the antiangiogenic,antimetastatic, anti-inflammatory, antimicrobial, anticoagulant and/orantithrombotic treatment of a mammalian subject. The compounds andcompositions thereof also have utility for lowering blood triglyceridelevels and inhibiting cardiovascular disease in a mammalian subject. Thecompounds additionally have utility in the prevention of the foregoingdisorders when administered to a mammalian subject.

BACKGROUND ART

The sulfated oligosaccharide agent known as PI-88 [1,2] (see compound 1below) has been shown to be a promising inhibitor of tumour growth andmetastasis [1,3] and is undergoing Phase II clinical trials in cancerpatients [4]. PI-88 exerts antiangiogenic effects by inhibiting theinteractions of angiogenic growth factors (principally FGF-1, FGF-2 andVEGF) and their receptors with heparan sulfate [1,5]. In addition, PI-88is a potent inhibitor of the enzyme heparanase, a glycosidase thatcleaves the heparan sulfate side chains of proteoglycans that are amajor constituent of the extracellular matrix (ECM) and basementmembranes surrounding tumour cells [1,2]. Heparanase has been stronglyimplicated in angiogenesis: it is able to liberate active heparansulfate-bound angiogenic growth factors from the ECM and is involved inthe degradation of the ECM and subsequent tissue remodeling associatedwith the sprouting of new blood vessels [6]. The degradation of the ECMby heparanase is also crucial in the spread of tumour cells (metastasis)by allowing them to pass into the blood stream and lodge in remote siteswhere they can form secondary tumours [6,7].

In addition to its antiangiogenic effects, PI-88 inhibits the bloodcoagulation cascade by (i) inhibiting proteases in the intrinsicpathway, (ii) stimulating the release of tissue factor pathway inhibitor(TFPI), and (iii) activating the heparin cofactor II-mediated inhibitionof thrombin. However, PI-88 does not interact with AT III and thus showsno anti-Xa or AT III-mediated anti-IIa activity [8,9].In vivo studies inmonkeys have shown that low doses of PI-88 stimulate release of allheparan sulfate bound TFPI from the vascular cell wall [9]. Apart fromits effect on coagulation, TFPI was recently shown to be anantiangiogenic agent [10] and an inhibitor of metastasis [11]. PI-88 hasalso been shown to block vascular smooth muscle cell proliferation andintimal thicking [12], to inhibit herpes simplex virus (HSV) infectionof cells and the cell-to-cell spread of HSV-1 and HSV-2 [13], and toinhibit proteinuria in passive Heymann nephritis [14].

PI-88 is a mixture of highly sulfated, monophosphorylated mannoseoligosaccharides ranging in size from di- to hexasaccharide [15,16].PI-88 is prepared by exhaustive sulfonation [2,16] of theoligosaccharide phosphate fraction (2) (see formula I following thisparagraph) obtained by mild, acid-catalyzed hydrolysis of theextracellular phosphomannan of the yeast Pichia (Hansenula) holstii NRRLY-2448 [17,18]. The major components are the penta- and tetrasaccharidephosphates 3 (˜60%) and 4 (˜30%), respectively, whilst the remaining 10%is made up of di-, tri- and hexasaccharide phosphates (5-7) and atetrasaccharylamine (not shown) [15,16]. Formula I

n R R¹ 1 0-4 SO₃Na or H PO₃Na₂ 2 0-4 H PO₃Na₂ 3 3 H PO₃Na₂ 4 2 H PO₃Na₂5 0 H PO₃Na₂ 6 1 H PO₃Na₂ 7 4 H PO₃Na₂ 8 0 H H 9 1 H H 10 2 H H 11 3 H H

Various other polysulfated oligo- and polysaccharides and theirderivatives are well known to exhibit similar types of biologicalactivities to PI-88 [19-25]. These biological activities are attributedto the inhibition of various heparan sulfate (HS)-binding proteins. Theobject of the present invention is to create derivatives of PI-88 thathave similar biological activities but with improved properties, forexample, in their pharmacokinetic and/or ADME (absorption, distribution,metabolism, excretion) profiles. A further object of the invention is toprovide compounds comprising a single carbon skeleton to facilitatetheir synthesis and characterization.

SUMMARY OF THE INVENTION

According to a first embodiment of the invention, there is provided acompound of the general formula:X—[Y]_(n)-Z-UR¹  IIwherein;

X, Y and Z are each a monosaccharide unit with a group UR bonded via asingle or multiple bond to each non-linking carbon of X, Y and Z, exceptcarbon-1 of monosaccharide Z which bears UR¹ bonded via a single ormultiple bond;

n is an integer having a value of 0-6;

each U is independently C, N, S or O or their higher oxidation states,including CO, COO, NO, NO₂, S(O), S(O)O;

each R is independently SO₃M or H, where M is any pharmaceuticallyacceptable cation or is any alkyl, aryl, acyl, aroyl, alkyl sulfonyl,aryl sulfonyl, PEG, a PEG derivative, H or the group

where independently in each AB group, A is O or NH, and B is H, or Mwhere M is as defined above, or an alkyl, aryl or any other suitablegroup;

R¹ is SO₃M, H, alkyl, aryl, acyl, aroyl, alkyl sulfonyl, aryl sulfonyl,PEG or a PEG derivative, or R¹ together with U is N₃ or a substitutedtriazole or derivative, or a substituted tetrazole or derivative, or asubstituted aryl or derivative, or a substituted heteroaryl orderivative;

with the proviso that when all UR¹ and UR groups are OSO₃M or OH(excluding the exocyclic methylene group of monosaccharide X), theexocyclic methylene group of monosaccharide X cannot be a OPO₃M₂ group.

According to a second embodiment of the invention, there is provided apharmaceutical or veterinary composition for the prevention or treatmentin a mammalian subject of a disorder resulting from angiogenesis,metastasis, inflammation, coagulation/thrombosis, raised bloodtriglyceride levels, microbial infection and/or cardiovascular disease,which composition comprises at least one compound according to the firstembodiment together with a pharmaceutically or veterinarially acceptablecarrier or diluent for said at least one compound.

A third embodiment of the invention comprises the use of a compoundaccording to the first embodiment in the manufacture of a medicament forthe prevention or treatment in a mammalian subject of a disorderresulting from angiogenesis, metastasis, inflammation,coagulation/thrombosis, raised blood triglyceride levels, microbialinfection and/or cardiovascular disease.

According to a fourth embodiment of the invention there is provided amethod for the prevention or treatment in a mammalian subject of adisorder resulting from angiogenesis, metastasis, inflammation,coagulation/thrombosis, raised blood triglyceride levels, microbialinfection and/or cardiovascular disease, which method comprisesadministering to the subject an effective amount of at least onecompound according to the first embodiment, or a composition comprisingsaid at least one compound.

A further embodiment of the invention comprises novel intermediates andthe synthetic pathway resulting in the sulfated oligosaccharides of thefirst embodiment.

Preferred compounds according to the invention, where the monosaccharidemolecules are exclusively D-mannose and the glycosidic linkages areα-(1→2) and α-(1→3), are depicted in the following structure:

where R, R¹, U and n are as defined above.

In order that the invention may be more readily understood and put intopractice, one or more preferred embodiments thereof will now bedescribed, by way of example only, with reference to the accompanyingfigure.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the effect of PI-88-like compounds on HSV-1 infectivity (A)and HSV-1 cell-to-cell spread (B). In panel A, the results are expressedas a percentage of the number of viral plaque forming units (PFU) formedin cells infected with the compound-treated virions relative tomock-treated controls. In panel B, the results are expressed as apercentage of the average area of 20 viral plaques formed in thecontinuous presence of compound relative to mock-treated control cells.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present inventors have found that a large number of sulfatedoligosaccharide derivatives can be synthesised using a number ofdifferent strategies as broadly described below and as illustrated inthe examples. These compounds have utility in the prevention ortreatment in mammalian subjects of a disorder resulting fromangiogenesis, metastasis, inflammation, coagulation, thrombosis,elevated blood triglyceride levels, microbial infection and/orcardiovascular disease. This utility results from the ability of thecompounds to block the binding of heparan sulfate-binding proteins totheir receptors, or to inhibit the activity of the enzyme heparanase.

With regard to the subject compounds of formula II, the monosaccharideunits X, Y and Z can be, for example, any hexose or pentose and can beeither a D or L isomer. Such hexoses include glucose, mannose, altrose,allose, talose, galactose, idose and gulose. Such pentoses includeribose, arabinose, xylose and lyxose. The glycosidic linkages of themonosaccharide units can be exclusively of one type or of differenttypes in terms of configuration and linkage.

The pharmaceutically acceptable cation M is preferably sodium.

With regard to integer n, a preferred value is 3 so as to provide acompound which is a pentasaccharide.

A preferred suitable R¹ group is n-octyl.

The anomeric configuration, where applicable, at UR¹ of compounds offormula II can be either α or β or an anomeric α/β mixture.

With regard to the substituents given above in the definition ofcompounds of formula II, the term “alkyl”, when used alone or incompound words such as “arylalkyl” refers to a straight chain, branchedor cyclic hydrocarbon group, preferably C₁₋₂₀, such as C₁₋₁₀. Forexample, the term “C₁-C₆ alkyl” refers to a straight chain, branched orcyclic alkyl group of 1 to 6 carbon atoms. Examples of “C₁₋₁₆ alkyl”include methyl, ethyl, iso-propyl, n-propyl, n-butyl, sec-butyl,t-butyl, n-pentyl, isopentyl, 2,2-dimethypropyl, n-hexyl,2-methylpentyl, 2,2-dimethylbutyl, 3-methylpentyl and 2,3-dimethypropyl,n-hexyl, 2-methylpentyl, 2,2-dimethylbutyl, 3-methylpentyl and2,3-dimethylbutyl. Examples of cyclic C₁₋₆ allyl include cyclopropyl,cyclobutyl, cyclopentyl and cyclohexyl Other examples of alkyl include:heptyl, 5-methylhexyl, 1-methylhexyl, 2,2-dimethylpentyl,3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl,1,3-dimethylpentyl, 1,4-dimethyl-pentyl, 1,2,3-trimethylbutyl,1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, octyl, 6-methylheptyl,1-methylheptyl, 1,1,3,3-tetramethylbutyl, nonyl, 1-, 2-, 3-, 4-, 5-6- or7-methyl-octyl, 1-, 2-, 3-, 4- or 5-ethylheptyl, 1-, 2- or3-propylhexyl, decyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- and 8-methylnonyl, 1-,2-, 3-, 4-, 5- or 6-ethyloctyl, 1-, 2-, 3- or 4-propylheptyl, undecyl,1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-methyldecyl, 1-, 2-, 3-, 4-, 5-, 6-or 7-ethylnonyl, 1-, 2-, 3-, 4- or 5-propylocytl, 1-, 2- or3-butylheptyl, 1-pentylhexyl, dodecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8,-9-or 10-methylundecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-ethyldecyl, 1-, 2-,3-, 4-, 5- or 6-propylnonyl, 1-, 2-, 3- or 4-butyloctyl,1-2-pentylheptyl and the like. An alkyl group may be optionallysubstituted by one or more optional substituents as herein defined.Optionally, the straight, branched or cyclic hydrocarbon group (havingat least 2 carbon atoms) may contain one, two or more degrees ofunsaturation so as to form an alkenyl or alkynyl group, preferably aC₂₋₂₀ alkenyl, more preferably a C₂₋₆ alkenyl, or a C₂₋₂₀ allynyl, morepreferably a C₂₋₆ alkynyl. Examples thereof include a hydrocarbonresidue containing one or two or more double bonds, or one or two ormore triple bonds. Thus, “alkyl” is taken to include alkenyl andalkynyl.

The term “aryl”, when used alone or in compound words such as“arylalkyl”, denotes single, polynuclear, conjugated or fused residuesof aromatic hydrocarbons or aromatic heterocyclic (heteroaryl) ringsystems, wherein one or more carbon atoms of a cyclic hydrocarbonresidue is substituted with a heteroatom to provide an aromatic residue.Where two or more carbon atoms are replaced, this may be by two or moreof the same heteroatom or by different heteroatoms. Suitable heteroatomsinclude O, N, S and Se.

Examples of “aryl” include phenyl, biphenyl, terphenyl, quaterphenyl,naphtyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl,benzanthracenyl, dibenzanthracenyl, phenanthrenyl, fluorenyl, pyrenyl,idenyl, azulenyl, chrysenyl, pyridyl, 4-phenylpyridyl, 3-phenylpyridyl,thienyl, furyl, pyrrolyl, indolyl, pyridazinyl, pyrazolyl, pyrazinyl,thiazolyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl,benzothienyl, purinyl, quinazolinyl, phenazinyl, acridinyl, benoxazolyl,benzothiazolyl and the like. Preferred hydrocarbon aryl groups includephenyl and naphthyl. Preferred heterocyclic aryl groups include pyridyl,thienyl, furyl, pyrrolyl. An aryl group may be optionally substituted byone or more optional substituents as herein defined.

The term “acyl” refers to a group —C(O)—R wherein R is an alkyl or arylgroup. Examples of acyl include straight chain or branched alkanoyl suchas acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl,2,2-dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl,decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl,pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyland icosanoyl; cycloalkylcarbonyl, such as cyclopropylcarbonylcyclobutylcarbonyl, cyclopentylcarbonyl and cyclohexylarbonyl; aroylsuch as benzoyl, toluoyl and naphthoyl; aralkanoyl such as phenylakanoyl(e.g. phenylaceyl, phenylpropanoyl, phenylbutanoyl, phenylisobutylyl,phenypentanoyl and phenylhexanoyl) and naphthylalkanoyl (e.g.naphthyhlacetyl, naphthylpropanoyl and naphthylbutanoyl). Since the Rgroup may be optionally substituted as described above, “acyl” is takento refer to optionally substituted acyl.

Optional substituents for alkyl, aryl or acyl include halo (bromo,fluoro, chloro, iodo), hydroxy, C₁₋₆alkyl (e.g. methyl, ethyl, propyl(n- and i-isomers)), C₁₋₆alkoxy (e.g. methoxy, ethoxy, propoxy (n- andi-isomers), butoxy (n-, sec- and t-isomers), nitro, amino,C₁₋₆alkylamino (e.g. methyl amino, ethyl amino, propyl (n- andi-isomers)amino), C₁₋₆dialkylamino (e.g. dimethylamino, diethylamino,diisopropylamino), halomethyl (e.g. trifluoromethyl, tribromomethyl,trichloromethyl), halomethoxy (eg trifluoromethoxy, tribromomethoxy,trichloromethoxy) and acetyl.

A 5-6 membered heterocyclyl group includes aromatic 5-6-memberedheterocyclic groups (heteroaryl) as described above and non aromatic5-6-membered heterocyclic groups containing one or more heteroatoms(preferably 1 or 2) independently selected from O, N, S and Se. Examplesthereof include dioxanyl, pyranyl, tetrahydrofuranyl, piperidyl,morpholino, piperazinyl, thiomorpholino and saccharides.

The degree of sulfation of compounds according to the invention istypically at least 50%. That is, at least 50% of the R groups of anoligosaccharide derivative comprise SO₃M. The degree of sulfation istypically from 70 to 100% and preferably is at least as high as 90%.

The PI-88 derivatives of formula II can be made via a stepwise syntheticroute or by starting with the PI-88 backbone already in place (using thereadily available compounds 8-11; see formula I above) and making thedesired modifications thereto. The inventors determined from aconsideration of the structure of PI-88 (1) and its precursor (2), thatthere are two preferred points of derivatisation: at the reducing end(A) and at the terminal 6-position at the non-reducing end (B) asillustrated in the following structural formula.

It should be noted that di-, tri-, tetra- and pentasaccharide (andlarger) derivatives all can be made by the same chemistry. However, thepentasaccharide derivatives are preferred since they are the mostbiologically active [1,2,5,8,13]. All the derivatives made are thensubject to deprotection (typically, deacetylation with NaOMe) and theresulting polyol sulfonated with a sulfonating reagent such as sulfurtrioxide pyridine complex or sulfur trioxide trimethylamine complex.

As indicated above, the compounds according to the invention haveutility in the prevention or treatment in mammalian subjects of adisorder resulting from angiogenesis, metastasis, inflammation,coagulation, thrombosis, elevated blood triglyceride levels, microbialinfection or cardiovascular disease. The compounds have particularutility in the treatment of the foregoing disorders in humans. Thecompounds are typically administered as a component of a pharmaceuticalcomposition as described in the following paragraphs. As will beillustrated below, the compounds show similar or superior activities toPI-88 itself.

Pharmaceutical compositions for oral administration can be in tablet,capsule, powder or liquid form. A tablet can include a solid carriersuch as gelatine or an adjuvant or an inert diluent. Liquidpharmaceutical compositions generally include a liquid carrier such aswater, petroleum, animal or vegetable oils, a mineral oil or a syntheticoil. Physiological saline solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol may be included. Suchcompositions and preparations will generally contain at least 0.1 wt %of the compound.

Parenteral administration includes administration by the followingroutes: intravenously, cutaneously or subcutaneously, nasally,intramuscularly, intraocularly, transepithelially, intraperitoneally andtopically. Topical administration includes dermal, ocular, rectal,nasal, as well as administration by inhalation or by aerosol means. Forintravenous, cutaneous or subcutaneous injection, or injection at a sitewhere treatment is desired, the active ingredient will be in the form ofa parenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of skill in the art willbe well able to prepare suitable solutions using, for example, solutionsof the subject compounds or derivatives thereof.

In addition to the at least one compound and a carrier or diluent,compositions according to the invention can further include apharmaceutically or veterinarially acceptable excipient, buffer,stabiliser, isotonicising agent, preservative or anti-oxidant or anyother material known to those of skill in the art. It will beappreciated by the person of skill that such materials should benon-toxic and should not interfere with the efficacy of the compound(s).The precise nature of any additive may depend on the route ofadministration of the composition: that is, whether the composition isto be administered orally or parenterally. With regard to buffers,aqueous compositions typically include such substances so as to maintainthe composition at a close to physiological pH or at least within arange of about pH 5.0 to 8.0.

Compositions according to the invention can also include activeingredients in addition to the at least one compound. Such ingredientswill be principally chosen for their efficacy as anti-angiogenic,anti-metastatic, anti-inflammatory, anti-coagulant, antimicrobial andantithrombotic agents, and agents effective against elevated bloodtriglyceride levels and cardiovascular disease, but can be chosen fortheir efficacy against any associated condition.

A pharmaceutical or veterinary composition according to the inventionwill be administered to a subject in either a prophylactically effectiveor a therapeutically effective amount as necessary for the particularsituation under consideration. The actual amount of at least onecompound administered by way of a composition, and rate and time-courseof administration, will depend on the nature and severity of thecondition being treated or the prophylaxis required. Prescription oftreatment such as decisions on dosage and the like will be within theskill of the medical practitioner or veterinarian responsible for thecare of the subject. Typically however, compositions for administrationto a human subject will include between about 0.01 and 100 mg of thecompound per kg of body weight and more preferably between about 0.1 and10 mg/kg of body weight.

The compounds can be included in compositions as pharmaceutically orveterinarially acceptable derivatives thereof. As used herein“derivatives” of the compounds includes salts, coordination complexeswith metal ions such as Mn²⁺ and Zn²⁺, esters such as in vivohydrolysable esters, free acids or bases, hydrates, or prodrugs.Compounds having acidic groups such as phosphates or sulfates can formsalts with alkaline or alkaline earth metals such as Na, K, Mg and Ca,and with organic amines such as triethylamine and Tris (2-hydroxyethyl)amine. Salts can also be formed between compounds with basic groups,such as amines, with inorganic acids such as hydrochloric acid,phosphoric acid or sulfuric acid, or organic acids such as acetic acid,citric acid, benzoic acid, fumaric acid, or tartaric acid. Compoundshaving both acidic and basic groups can form internal salts.

Esters can be formed between hydroxyl or carboxylic acid groups presentin the compound and an appropriate carboxylic acid or alcohol reactionpartner, using techniques that will be well known to those of skill inthe art.

Prodrug derivatives of the compounds of the invention can be transformedin vivo or in vitro into the parent compounds. Typically, at least oneof the biological activities of a parent compound may be suppressed inthe prodrug form of the compound, and can be activated by conversion ofthe prodrug to the parent compound or a metabolite thereof. Examples ofprodrugs are glycolipid derivatives in which one or more lipid moietiesare provided as substituents on the moieties, leading to the release ofthe free form of the compound by cleavage with an enzyme havingphospholipase activity. Prodrugs of compounds of the invention includethe use of protecting groups which may be removed in vivo to release theactive compound or serve to inhibit clearance of the drug. Suitableprotecting groups will be known to those of skill in the art and includean acetate group.

As also indicated above, compounds according to the invention haveutility in the manufacture of a medicament for the prevention ortreatment in a mammalian subject of a disorder resulting fromangiogenesis, metastasis, inflammation, coagulation/thrombosis,microbial infection, elevated blood triglyceride levels and/orcardiovascular disease. Processes for the manufacture of suchmedicaments will be known to those of skill in the art and include theprocesses used to manufacture the pharmaceutical compositions describedabove.

A general description of the synthetic routes to the compounds accordingto the invention will now be given. For simplicity, in all schemes,figures and tables which follow, R¹ will represent an α-(1→3)-linkedMan₄ tetrasaccharide portion (with or without a terminal 6-O-phosphogroup), unless otherwise indicated.

General Procedures

Glycoside Derivatives of PI-88 (0-, S- and C-Glycosides)

Glycoside derivatives can be readily prepared by activating theoligosaccharide (with or without a terminal 6-O-phospho group) forglycosylation and condensing it with an appropriate alcohol. A suitablemethod is the Lewis acid-catalysed or promoted reaction of aperacetylated sugar, e.g, 12, with an alcohol acceptor, e.g. to give 13and 14. Where a more unreactive acceptor is required, a more reactiveglycosyl donor needs to be prepared, e.g, the trichloroacetimidate 15 isused to prepare the PEGylated derivatives 16 and 17 (Scheme 1).

Various other types of donors are known in the art and are suitable asdonors, e.g., thioglycosides, halides, n-pentenyl glycosides,selenoglycosides etc. Those skilled in the art will recognize that S-and C-glycosides can be prepared by similar or related methods known inthe literature, for example by using an appropriate thiol (or thiolderivative) or a known carbon nucleophile (e.g., allyltrimethylsilane oran appropriate phenol) with a suitably activated donor. The product canthen easily be deacetylated and sulfonated. The product of theglycosylation may be a single anomer (α or β) or a mixture of bothanomers. Both the pure α and β anomers and the anomeric mixture aresuitable for subsequent transformations. This also applies to otherderivatives obtained through manipulation of the anomeric centredescribed in subsequent sections. Therefore, where a single anomer isdenoted it is implied that the opposite anomer or a mixture of the twoanomers is also claimed. It will also be clear to those skilled in theart that the initially formed glycoside, depending on the nature of theaglycone, can be further derivatized. As an example, if one uses2-bromohexanol as the alcohol, the product can be converted into anazide (18). This is an extremely versatile compound (Scheme 2) and mayfurther functionalized by, for example, cycloaddition with a compoundcontaining a suitable dipolarophile. Alternatively, the azide can bereduced to an amine and then further functionalized, for example, byalkylation, acylation, a 4-component Ugi condensation etc.

N-Linked Derivatives

From 12, Lewis acid catalysed reaction with TMSN₃ leads to the azide 19(predominantly α). Alternatively, the β-azide 20 can be formedexclusively by initial formation of the α-bromide followed bydisplacement with NaN₃ (Scheme 3). The bromide can also be used as anintermediate for the preparation of thioglycosides or isothiocyanates,for example. The azides can be deprotected and sulfonated as is, or canbe reduced and acylated with various acid chlorides to provide a seriesof glycosyl amides (Scheme 3).

Non-Reducing End Derivatives

Derivatization can also be accomplished at the non-reducing end, forexample, by the use of phosphorylated oligosaccharides (eitherindividually or as a mixture) and derivatizing through the phosphategroup, e.g., preparation of phosphate esters or phosphoramides. Indeed,suitable compounds can be prepared whereby the reducing end is alsoderivatized, with either a similar or different functional group.

Having broadly described the invention, non-limiting examples of thecompounds, their synthesis, and their biological activities, will now begiven.

EXAMPLES

Neutral Manno-Oligosaccharides

(a) The manno-oligosaccharides (8) α-D-Man-(1→2)-D-Man, (9)α-D-Man-(1→3)-α-D-Man-(1→2) -D-Man, (10)α-D-Man-(1→3)-α-D-Man-(1→3)-α-D-Man-(1→2)-D-Man, and (11)α-D-Man-(1→3)-α-D-Man-(1→3)-α-D-Man-(1→3)-α-D-Man-(1→2)-D-Man, wereisolated from the neutral fraction of the mild acid-catalysed hydrolysisof the extracellular phosphomannan from P. holstii NRRL Y-2448 by sizeexclusion chromatography according to the literature procedure [17].Alternatively, the oligosaccharides 8-11 were synthesized in a stepwisemanner from monosaccharide building blocks as described in example 1(see below).

(b) Alternatively, the neutral fraction was directly acetylated (excessAc₂O/pyridine) and the individual peracetylated oligosaccharidesisolated by flash chromatography (silica gel) and used in this formdirectly in the next step.

(c) In another approach, the peracetylated mixture from (b) was useddirectly in the next step and the individual products were then isolatedby flash chromatography.

General Procedure for Deacetylation

A solution of the peracetate in anhydrous methanol (0.1 M) was treatedwith a solution of sodium methoxide in methanol (1.35 M, 0.2-0.6 eq).The mixture was stirred at room temperature for 1-3 h (monitored byTLC). Acidic resin AG®-50W-X8 (H⁺ form) was added to adjust pH=6-7, themixture was filtered and the resin was rinsed with methanol. Thecombined filtrate and washings were concentrated in vacuo and thoroughlydried to give the polyol product.

General Procedure for Sulfonation

A mixture of the polyol and SO₃.trimethylamine or SO₃.pyridine complex(2 eq. per alcohol) in DMF was heated (60° C., o/n). The cooled (r.t.)reaction mixture was treated with MeOH and then made basic (to pH>10) bythe addition of Na₂CO₃ (10% w/w). The mixture was filtered and thefiltrate evaporated and co-evaporated (H₂O). The crude polysulfatedmaterial was dissolved in H₂O and subjected to size exclusionchromatography (see below) to yield the sulfated product. When required,after lyophilisation the product was passed through an ion-exchangeresin column (AG®-50W-X8, Na⁺ form, 1×4 cm, deionized H₂O, 15 mL) inorder to transfer the product uniformly into the sodium salt form. Thesolution collected was evaporated and lyophilised to give the finalproduct as a colourless glass or white power.

Size Exclusion Chromatography

Size exclusion chromatography was performed over Bio-Gel P-2 in a 5×100cm column and a flow rate of 2.8 mL/min of 0.1 M NH₄ ⁺.HCO₃ ⁻,collecting 2.8 min (7.8 mL) fractions. Fractions were analysed forcarbohydrate content by spotting onto silica gel plates andvisualisation by charring, and/or analysed for poly-charged species bythe dimethyl methylene blue test. Finally, fractions were checked forpurity by CE¹⁵ and those deemed to be free of salt were pooled andlyophilised. In the cases of the presence of undersulfated by-productsor other organic salt contaminants (normally only small amounts, butquite often detected), an LH20 column chromatography (2×95 cm, deionizedwater, 1.2 mL/min, 3.5 min per vial) was applied to remove themcompletely.

Example 1 Total Synthesis of Neutral Manno-Oligosaccharides (8-11) fromPichia

Benzyl2-O-(3-O-Allyl-2,4,6-tri-O-benzoyl-α-D-mannopyranosyl)-3,4,6-tri-O-benzyl-α-D-mannopyranoside(24)

A mixture of 3-O-allyl-2,4,6-tri-O-benzoyl-α-D-mannopyranosyltrichloroacetimidate [26] (902 mg, 1.21 mmol) and benzyl3,4,6-tri-O-benzyl-α-D-mannopyranoside [27] (723 mg, 1.34 mmol) in1,2-DCE (10 mL), was stirred in the presence of mol. sieves (1.0 g of 3Å powder) under an atmosphere of argon (30 min). The mixture was cooled(0° C.) with continued stirring (10 min) prior to the addition of TMSOTf(219 μL, 1.21 mmol). After some time (10 min), Et₃N (100 μL) wasintroduced and the mixture was filtered. The solvent was evaporated andthe residue subjected to FC (10-50% EtOAc/hexane) to yield thetribenzoate (24) as, a colourless oil (1.14 g, 84%). ¹HNMR (CDCl₃) δ3.67-3.81, 3.88-3.95, 4.06-4.15, 4.30-4.35 (4 m, 12H; H-2^(I),-3^(I),-4^(I),-5^(I),-6a^(I),-6b^(I),-3^(II),-5^(II),-6a^(II),-6b^(II),OCH₂), 4.94-4.70 (m, 7H; CH₂Ph), 4.84 (d, 1H, J_(A,B) 10.8 Hz; A of ABquartet), 4.93-4.96, 5.04-5.09 (2 m, 2H; ═CH₂), 5.02 (d, 1H, J_(1,2) 1.9Hz; H-1^(I)), 5.24 (d, 1H; J_(1,2) 1.9 Ha; H-1^(II)), 5.59-5.69 (m, 1H;═CH), 5.72 (dd, 1H, J_(2,3) 3.1 Hz; H-2^(II)), 5.75 (dd, 1H, J_(3,4)9.8, J_(4,5) 9.9 Hz; H-4^(II)), 7.09-7.58, 7.97-8.06 (2 m, 35H; Ar).¹³CNMR (CDCl₃) δ 61.50, 63.49 (2 C; C-6^(I), -6^(II)), 68.63, 69.17,69.31, 69.46, 69.64, 71.08, 72.04, 72.64, 73.60, 74.73, 75.30, 75.38 (13C; C-3^(I),-4^(I),-5^(I),-2^(II),-3^(II),-4^(II),-5^(II), OCH₂, CH₂Ph),79.97 (C-2^(I)), 98.52, 99.60 (C-1^(I),-1^(II)), 117.67 (═CH₂),127.70-138.43 (43 C; ═CH, Ar), 165.61, 165.69, 166.42 (3 C; C═O).

Benzyl2-O-(2,4,6-Tri-O-benzoyl-α-D-mannopyranosyl)-3,4,6-tri-O-benzyl-α-D-mannopyranoside(25)

PdCl₂ (40 mg) was added to a solution of the allyl ether (24) (1.09 g,0.97 mmol) in MeOH (10 mL) and 1,2-DCE (10 mL) and the combined mixturewas heated (70°, 40 min). After the time, the solvents were evaporatedand the residue subjected to FC (20-30% EtOAc/hexanes) to yield thealcohol (25) as a colourless oil (0.96 g, 91%). ¹H NMR (CDCl₃) δ3.68-3.81, 3.97-4.06, 4.32-4.71 (3 m, 18H;H-2^(I),-3^(I),-4^(I),-5^(I),-6a^(I),-6b^(I),-3^(II),-5^(II),-6a^(II),-6b^(II),CH₂Ph), 4.84 (d, 1H, J_(A,B) 12 Hz; A of AB quartet), 5.05 (d, 1H,J_(1,2) 1.9 Hz; H-1^(I)), 5.26 (d, 1H; J_(1,2) 1.9 Ha; H-1^(II)), 5.61(dd, 1H, J_(2,3) 3.3 Hz; H-2^(II)), 5.67 (dd, 1H, J_(3,4) 9.8, J_(4,5)9.9 Hz; H-4^(II)), 7.13-7.40, 7.48-7.59, 7.98-8.06 (3 m, 35H; Ar). ¹³CNMR (CDCl₃) δ 60.61, 63.32 (2 C; C-6^(I),-6^(II)), 69.06, 69.12, 69.25,69.44, 70.45, 72.14, 72.65, 72.77, 73.48, 74.79, 75.48, 75.47, 76.23 (13C; C-3^(I),-4^(I),-5^(I),-2^(II),-3^(II),-4^(II),-5^(II), OCH₂, CH₂Ph),79.66 (C-2^(I)), 98.34, 99.40 (C-1^(I),-1^(II)), 127.70-138.47 (42 C;Ar), 165.97, 166.36, 166.97 (3 C; C═O).

Benzyl2-O-[(3-O-Allyl-2,4,6-tri-O-benzoyl-α-D-mannopyranosyl)-(1→3)-(2,4,6-tri-O-benzoyl-α-D-mannopyranosyl)]-3,4,6-tri-O-benzyl-α-D-mannopyranoside(26)

A mixture of 3-O-allyl-2,4,6-tri-O-benzoyl-α-D-mannopyranosyltrichloroacetimidate (742 mg, 1.01 mmol) and the alcohol (25) (908 mg,0.84 mmol) in 1,2-DCE (10 mL), was stirred in the presence of mol.sieves (1.0 g of 3 Å powder) under an atmosphere of argon (30 min). Themixture was cooled (0° C.) with continued stirring (10 min) prior to theaddition of TMSOTf (181 μL, 1.01 mmol). After some time (10 min), Et₃N(100 μL) was introduced and the mixture was filtered. The solvent wasevaporated and the residue subjected to FC (10-50% EtOAc/hexane) toyield the hexabenzoate (26) as, a colourless oil (1.26 g, 90%). ¹H NMR(CDCl₃) δ 3.51-3.56, 3.66-4.06, 4.23-4.27, 4.30-42, 4.47-4.72, 4.78-4.86(6 m, 26H;H-2^(I),-3^(I),-4^(I),-5^(I),-6a^(I),-6b^(I),-3^(II),-5^(II),-6a^(II),-6b^(II),-3^(III),-5^(III),-6a^(III),-6b^(III), OCH₂, ═CH₂, CH₂Ph), 5.04 (d, 1H, J_(1,2) 1.7 Hz;H-1^(I)), 5.15 (dd, 1H, J_(1,2) 1.8, J_(2,3) 2.7 Hz; H-2^(II)), 5.26 (d,1H; H-1^(II)), 5.28 (d, 1H, J_(1,2) 1.7 Hz; H-1^(III)), 5.33-5.43 (m,1H; ═CH), 5.77-5.82 (m, 2H; H-4^(II),-2^(III)), 5.92 (dd, 1H, J_(3,4)9.5, J_(4,5) 9.8 Hz; H-4^(III)), 7.00-7.61, 7.80-8.19 (2 m, 50H; Ar).

Benzyl2-O-[(2,4,6-Tri-O-benzoyl-α-D-mannopyranosyl)-(1→3)-(2,4,6-tri-O-benzoyl-α-D-mannopyranosyl)]-3,4,6-tri-O-benzyl-α-D-mannopyranoside(27)

PdCl₂ (40 mg) was added to a solution of the allyl ether (26) (394 mg,241 μmol) in MeOH (10 mL) and 1,2-DCE (10 mL) and the combined mixturewas heated (70°, 60 min). After the time, the solvents were evaporatedand the residue subjected to FC (20-30% EtOAc/hexanes) to yield thealcohol (27) as a colourless oil (317 mg, 84%). ¹H NMR (CDCl₃) δ3.67-3.82, 3.91-3.99, 4.01-4.21, 4.29-4.71 (4 m, 21H;H-2^(I),-3^(I),-4^(I),-5^(I),-6a^(I),-6b^(I),-3^(II),-5^(II)-,6a^(II),-6b^(II),-3 ^(III),5 ^(III), -6a^(III),-6b^(III)CH₂Ph), 4.83 (d, 1H,J_(A,B) 10.9 Hz; A of AB quartet), 5.03-5.05 (m, 2H; H-1^(I),-2^(II)),5.25-5.28 (m, 2H; H-1^(II),-1^(III)), 5.63 (dd, 1H, J_(3,4)=J_(4,5) 9.9Hz; H-4^(II)), 5.77 (dd, 1H, J_(1,2) 2.0, J_(2,3) 3.1 Hz; H-2^(III)),5.92 (dd, 1H, J_(3,4) 9.7, J_(4,5) 9.9 Hz; H-4^(III)), 6.99-7.62,7.80-8.16 (2 m, 50H; Ar).

Benzyl2-O-[(3-O-Allyl-2,4,6-Tri-O-benzoyl-α-D-mannopyranosyl)-(1→3)-(2,4,6-tri-O-benzoyl-α-D-mannopyranosyl)]-(1→3)-(2,4,6-tri-O-benzoyl-α-D-mannopyranosyl)]-3,4,6-tri-O-benzyl-α-D-mannopyranoside(28)

A mixture of 3-O-allyl-2,4,6-tri-O-benzoyl-α-D-mannopyranosyltrichloroacetimidate (102 mg, 138 μmol) and the alcohol (27) (135 mg,86.5 μmol) in 1,2-DCE (6 mL), was stirred in the presence of mol. sieves(100 mg of 3 Å powder) under an atmosphere of argon (30 min). Themixture was cooled (0°) with continued stirring (10 min) prior to theaddition of TMSOTf (25 μL, 138 μmol). After some time (10 min), Et₃N(100 μL) was introduced and the mixture was filtered. The solvent wasevaporated and the residue subjected to FC (10-50% EtOAc/hexane) toyield the nonabenzoate (28) as, a colourless oil (173 mg, 94%). ¹H NMR(CDCl₃) δ 3.44-3.49, 3.60-3.99, 4.05-4.16, 4.42-4.44, 4.48-4.68,4.73-4.77 (6 m, 30H;

H-2^(I),-3^(I),-4^(I),-5^(I),-6a^(I),-6b^(I),-3^(II),-5^(II),-6a^(II),-6b^(II),-3^(III),5^(III),-6a^(III),-6b^(III),-3^(IV),-5^(IV),-6a^(IV),-6b^(IV), OCH₂,═CH₂, CH₂Ph), 4.83 (d, 1H, J_(A,B) 10.9 Hz; A of AB quartet), 5.01-5.04(m, 2H; H-1^(I),-2^(III)), 5.19-5.23 (m, 1H; H-2^(II)), 5.27-5.40 (m,4H; H-1^(I),-1^(II),-1^(III), ═CH₂), 5.61 (dd, 1H, J_(3,4)=_(4,5) 9.9Hz; H-4^(IV)), 5.77 (dd, 1H, J_(1,2) 2.0, J_(2,3) 3.1 Hz; H-2^(IV)),5.90-5.96 (m, 2H; H-4^(II),-4^(III)), 7.01-7.56, 770-8.16 (2 m, 65H;Ar).

Benzyl2-O-[(2,4,6-Tri-O-benzoyl-α-D-mannopyranosyl)-(1→3)-(2,4,6-tri-O-benzoyl-α-D-mannopyranosyl)]-(1→3)-(2,4,6-tri-O-benzoyl-α-D-mannopyranosyl)]-3,4,6-tri-O-benzyl-α-D-mannopyranoside(29)

PdCl₂ (30 mg) was added to a solution of the allyl ether (28) (155 mg,70.4 μmol) in MeOH (5 mL) and 1,2-DCE (5 mL) and the combined mixturewas heated (70°, 40 min). After this time, the solvents were evaporatedand the residue subjected to FC (20-40% EtOAc/hexanes) to yield thealcohol (29) as a colourless oil (97 mg, 64%). ¹H NMR (CDCl₃) δ3.67-3.82, 3.90-4.10, 4.24-4.68 (3 m, 26H;H-2^(I),-3^(I),-4^(I),-5^(I),-6a^(I),-6b^(I),-3^(II),-5^(II),-6a^(II),-6b^(II),-3^(III),-5^(III),-6a^(III),-6b^(III),-3^(IV),-5^(IV) ,-6a^(IV),-6b^(IV), CH₂Ph),4.84 (d, 1H, J_(A,B) 11.2 Hz; A of AB quartet), 4.86 (d, J_(1,2) 1.8 Hz;H-1^(I)), 4.90 (dd, 1H; J_(1,2) 1.8, J_(2,3) 3.1 Hz; H-2^(III)), 5.03(d, 1H, J_(1,2) 1.5 Hz; H-1^(IV)), 5.22 (dd, 1H, J_(1,2) 2.1, J_(2,3)2.6 Hz; H-2^(II)), 5.27-5.29 (m, 2H; H-1^(III),-1 ^(IV)), 5.46 (dd, 1H,J_(3,4) 9.7, J_(4,5) 9.9 Hz; H-4^(IV)), 5.79 (dd, 1H, J_(2,3) 2.9 Hz;H-2^(IV)), 5.90-5.96 (m, 2H; H-4^(II),-4^(III)), 7.01-7.56, 7.68-8.16 (2m, 65H; Ar).

Benzyl2-O-[(2,3,4,6-Tetra-O-Acetyl-α-D-mannopyranosyl)-(1→3)-(2,4,6-tri-O-benzoyl-α-D-mannopyranosyl)]-(1→3)-(2,4,6-tri-O-benzoyl-α-D-mannopyranosyl)-(1→3)-(2,4,6-tri-O-benzoyl-α-D-mannopyranosyl)]-3,4,6-tri-O-benzyl-α-D-mannopyranoside(30)

A mixture of 2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyltrichloroacetimidate [28] (39 mg, 78 μmol) and the alcohol (29) (85 mg,39 μmol) in 1,2-DCE (3 mL), was stirred in the presence of mol. sieves(100 mg of 3 Å powder) under an atmosphere of argon (30 min). Themixture was cooled (0°) with continued stirring (10 min) prior to theaddition of TMSOTf (14.2 μL, 78 μmol). After some time (30 min), Et₃N(100 μL) was introduced and the mixture was filtered. The solvent wasevaporated and the residue subjected to FC (30-60% EtOAc/hexane) toyield the tetraacetate (30) as, a colourless oil (85 mg, 87%). ¹H NMR(CDCl₃) δ 1.82-2.04 (4 s, 3H each; CH₃CO), 3.67-3.95, 4.05-4.72,4.82-5.03, 5.21-5.28, 5.69-5.50 (m, 43H; H-1^(I-IV),-2^(I-IV),-3^(I-IV), 4^(I-IV),-5 ^(I-IV), -6ab^(I-IV), CH₂Ph),7.01-7.56, 7.68-8.16 (2 m, 65H; Ar).

General Procedure for Deprotection of the Mannooligosaccharides (25, 27,29, 30)

(A) A small piece of sodium was added to a solution of the tetrabenzylether (25, 27, 29, 30) in MeOH and THF and the combined mixture wasstirred (r.t., o/n). After this time, the mixture was neutralised withDowex 50×8 resin (H⁺) form and filtered. The solvent was evaporated andco-evaporated (MeOH) and used in the following reaction without furtherpurification.

(B) Pd(OH)₂ (10% on C) was added to a solution of the crude product from(A) in THF and H₂O containing a little AcOH (500 □L) and the combinedmixture was vigorously stirred under hydrogen (100 p.s.i., 3 h). Afterthis time, the mixture was filtered and the solvent evaporated. Theresidue was subjected to gel flitration chromatography (Biogel P2; H₂O;60 ml/hr) to yield, after lyophilisation, the mannooligosaccharide(8-11) as a colourless powder. Compounds 8-11 were identical in allrespects to those isolated from the Pichia hydolysis as described above.

Example 2 Benzyl Glycoside Polysulfate (PG500)

Peracetate 12

The pentasaccharide 11 (1.03 g, 95% M5), sodium acetate (1.2 g) andacetic anhydride (50 mL) were heated, with stirring, at 140° C.overnight under a drying tube. The mixture was cooled to roomtemperature, evaporated to dryness, taken up in EtOAc, washed with brine(×3) and subjected to flash chromatography (40 g silica gel, 80:20EtOAc:Hx) to yield 810 mg of peracetate 12 as a glass along with lesspure material. ¹H NMR (400 MHz, CDCl₃) δ 6.14 (d, 0.84H, J=2.0,αH1^(I)), 5.71 (d, 0.16H, J=0.9, βH1^(I)), 5.30-5.10 (m, 8H), 5.00-4.85(m, 7H), 4.25-3.70 (m, 19H), 2.20-1.90 (m, 51H). HRMS calcd forC₆₄H₈₇O₄₃ [M+H]⁺1543.4623, found 1543.4599.

General Procedure for Direct Glycosylation of PeracetylatedOligosaccharides:

To a solution of the peracetate (eg, 12) (1 eq) in 3 Å MS dried DCM(0.03 M) was added the alcohol (6 eq). In some cases, small amount ofpowdered 3 Å MS was added. Boron trifluoride etherate (4 eq) was addedand the mixture was stirred under an atmosphere of argon at 60° C. or75° C. for 2 to 26 h. The mixture was cooled and triethylamine wasadded. The mixture was diluted with dichloromethane, washed with sat.aq. sodium carbonate and dried (anh. MgSO₄). The dried solution wasfiltered and the cake washed with dichloromethane. The combined filtrateand washings were concentrated, loaded on silica gel and purified byflash chromatography (silica, gradient elution with hexane-EtOAc 6:1 to1:4) to afford the desired glycoside after evaporation and drying underhigh vacuum.

Benzyl Glycoside 13

The glycosylation was performed using 12 and benzyl alcohol to give theproduct (13) as a colourless gum, 108 mg, 46% (Rf=0.32,hexane-EtOAc=1:3). ¹H NMR (CDCl₃, 400 MHz) δ 7.35-7.27 (m, 5H, C₆H₅),5.30-5.12 (m, 8H), 5.00-4.85 (m, 8H), 4.68 (AB quartet, 1H, J=11.8) and4.50 (AB quartet, 1H, J=11.8, PhCH₂O), 4.27-3.74 (m, 19H), 2.14(4),2.13(5), 2.13, 2.10, 2.08(4), 2.07(9), 2.07(6), 2.06(9), 2.06(6), 2.06(2×), 2.02, 2.00, 1.99, 1.97, 1.94 (15s, 48H, 16×Ac); ¹³C NMR (CDCl₃,100 MHz) δ 171.0, 170.5(3), 170.5(1), 170.5(0), 170.4, 170.3, 170.2,170.0(4), 170.0(2), 169.8(9), 169.8(8), 169.7, 169.6, 169.5(6), 169.4(6)and 169.3 (total 16×CO), 136.1 (ipso-C₆H₅), 128.5, 128.2 and 127.9 (o,m, p-C₆H₅), 99.2 (2C), 98.9, 98.8, 97.3 (5× sugar-C1), 76.7, 75.1,74.9(9), 74.9(7), 71.1, 70.9, 70.8, 70.2, 69.7, 69.5(9), 69.5(6),69.4(2), 69.3(7), 69.2, 68.6, 68.3, 67.1, 66.7(3), 66.6(7), 66.1, 65.5,62.4, 62.1, 61.9, 61.6 and 60.2 (26C, 25× sugar carbons excluding 5×sugar-C1 and benzyl CH₂), 20.9, 20.8(2), 20.8(0), 20.7(8), 20.7, 20.6,20.5(4), 20.5(1), 20.4 (9) and 20.4 (6) (10C, 16×Ac).

Benzyl Glycoside Polysulfate (PG500)

Compound 13 was deacetylated (HRMS calcd for polyol C₃₇H₅₉O₂₆ [M+H]⁺919.3296, found 919.3279) and sulfonated according to the generalprocedures to give the product (PG500) as a white powder, 76.1 mg, 44%.¹H NMR (D₂O, 400 MHz) δ 7.35-7.26 (m, 5H, C₆H₅), 5.32 (s, 1H), 5.30 (d,1H, J=1.2), 5.26 (d, 1H, J=2.0), 5.24 (d, 1H, J=1.6), 5.05 (dd, 1H,J=2.8, 2.0), 5.00 (d, 1H, J=2.0), 4.87-4.85 (m, 2H), 4.68-4.34 (m, 12H),4.32-3.86 (m, 17H); ¹³C NMR (D₂O, 100 MHz) δ 137.0, 129.5, 129.4, 129.1,100.5(9), 100.5(6), 100.2, 97.9, 93.8, 76.9, 76.8, 75.6, 75.5(3),75.4(8), 74.4, 73.8, 73.1, 73.0, 72.8, 72.7, 71.8, 71.3, 70.7, 70.6,70.4, 69.9, 69.8, 69.7, 68.0, 67.8, 67.5, 66.6, 66.3(7), 66.3(5).

Example 3 Octyl Glycoside Polysulfate (PG501)

Octyl Glycoside 14

The glycosylation was performed using 12 and octanol to give the product(14) as a colourless gum, 207 mg, 66% (Rf=0.41, hexane-EtOAc=1:3). ¹HNMR (CDCl₃, 400 MHz) δ 5.23-5.09 (m, 8H), 4.96-4.82 (m, 8H), 4.23-3.71(m, 19H), 3.59 (dt, 1H, J=9.4, 6.8, OCH₂R), 3.35 (dt, 1H, J=9.4, 6.8,OCH₂R), 2.11, 2.10(2), 2.09(8), 2.06, 2.05, 2.04(4), 2.04(1), 2.03(8),2.03, 2.02, 2.01, 1.99(3), 1.98(8), 1.96, 1.94 and 1.90 (16s, 48H,16×Ac), 1.52 (quintet, 2H, J=7.2, CH₂), 1.27-1.18 (m, 10H, (CH₂)₅), 0.80(t, 3H, J=7.2, CH₃); ¹³C NMR (CDCl₃, 100 MHz) δ 170.4(0) (2C), 170.3(8)(2C), 170.3, 170.2, 170.1, 169.9 (2C), 169.8(2), 169.7(5), 169.6, 169.5,169.4(4), 169.3(5), 169.3 (16×CO, 3 overlapped), 99.1 (2C), 98.8, 98.7,98.0 (5× sugar-C1), 77.0, 75.0, 74.8(3), 74.7(5), 71.0, 70.8, 70.7,70.1, 69.4(9), 69.4(7), 69.3(0), 69.2(7), 69.2, 68.3, 68.2(0), 68.1(6),67.2, 66.6(4), 66.6(0), 66.1, 65.4, 62.4, 62.3, 61.8 and 61.5 (25C,sugar carbons excluding sugar-C1 and octyl-CH₂O), 31.5, 29.1, 29.0,28.9, 25.9, 22.4 (6× octyl-CH₂), 20.7(3), 20.7(0), 20.6(7), 20.6, 20.5,20.4(3), 20.4(0), 20.3(9), 20.3(7) (9C, 16×Ac), 13.85 (octyl-CH₃).

Compound 14 was deacetylated (HRMS calcd for polyol C₃₈H₆₉O₂₆ [M+H]⁺941.40784, found 941.4060.) and sulfonated according to the generalprocedures to give the product (PG501) as a white powder, 195 mg, 72%.¹H NMR (D₂O, 400 MHz) δ 5.33 (s, 1H), 5.29 (d, 1H, J=1.6), 5.24 (d, 1H,J=1.6), 5.21 (d, 1H, J=1.6), 5.03 (dd, 1H, J=2.8, 2.0), 4.87 (d, 1H,J=1.6), 4.86-4.83 (m, 2H), 4.70-3.92 (m, 27H), 3.59 (dt, 1H, J=9.6,7.0), 3.44 (dt, 1H, J=9.6, 7.0), 1.48-1.40 (m, 2H), 1.21-1.08 (m, 10H),0.678 (t, 3H, J=7.2); ¹³C NMR (D₂O, 100 MHz) δ 100.5, 100.4, 100.1,100.0, 99.0, 98.4(1), 98.3(8), 98.3(6), 98.3(5), 76.8(5), 76.7(9), 76.7,76.6, 76.5(2), 76.4(7), 76.0, 75.4(O), 75.3(5), 75.3, 75.2, 74.3,73.0(5), 72.9(9), 72.7, 72.6, 71.7, 70.4, 70.2, 69.8(4), 69.7(5), 69.6,69.1, 67.8(5), 67.7(7), 66.5, 66.2, 31.5, 30.0, 28.8, 25.8, 22.5, 14.0.

Example 4 PEG₅₀₀₀ Polysulfate (PG504)

Imidate 15

(A) A mixture of the acetate (12) (68 mg, 51 μmol) and BnNH₂ (17 μL, 152μmol) in THF (2 mL), was stirred (r.t.) during some time (2 d). Themixture was diluted with CHCl₃ (20 mL) and subjected to work-up. Theorganic phase was evaporated and co-evaporated (2×10 mL MeCN) and usedin the following reaction without further purification.

(B) DBU (10 μL, 6.7 μmol) was added to a solution of the crude product(from A) and trichloroacetonitrile (1.0 mL, 10 mmol) in 1,2-DCE (4 mL)and the combined mixture was stirred (0° C.→12° C., o/n). The mixturewas concentrated and the residue subjected to FC (50-90% EtOAc/hexanes)to yield 15 as a pale yellow coloured oil (35 mg, 48%, 2 steps). ¹H NMR(400 MHz, CDCl₃) δ 8.70 (s, 1H, NH), 6.32 (d, 1H, J=2.0, H1^(I)),5.36-5.13 (m, 8H), 5.00-4.90 (m, 6H), 4.26-3.75 (m, 20H), 2.15-1.94 (m,48H).

PEG₅₀₀₀ Polysulfate (PG504)

(A) A mixture of the imidate 15 (33 mg, 20.2 μmol) andPEG₅₀₀₀-monomethyl ether (151 mg, 30.3 μmol) in 1,2-DCE (3 mL), wasstirred in the presence of mol. sieves (50 mg of 3 Å powder) under anatmosphere of argon (10 min). The mixture was cooled (−20° C.) withcontinued stirring (10 min) prior to the addition of TMSOTf (5 μL, 2.8μmol). After some time (20 min), Et₃N (10 μL) was introduced and themixture was filtered. The solvent was evaporated and the residuesubjected to FC (0-7.5% MeOH/CHCl₃) to yield 16 as a colourless glass(104 mg, 80%, based on average M_(r) 6483). ¹H NMR (400 MHz, CDCl₃) δ5.28-4.87 (m, 14H), 4.43-3.42 (m, 829H,), 3.34 (s, 3H, OMe), 2.15-1.94(m, 48H).

(B) Compound 16 (104 mg, 16 μmol) was deacetylated according to thegeneral procedure to yield Man₅-PEG₅₀₀₀-OMe as a colourless wax (82 mg,89%, based on average M_(r) 5769).

(C) The M₅-PEG₅₀₀₀-OMe (82 mg, 14 μmol) was sulfonated according to thegeneral procedure to yield PG504 as a colourless foam (45 mg, 42%, basedon average M_(r) 7401). ¹H NMR (400 MHz, D₂O) δ 5.34-4.87 (m, 7H),4.71-3.97 (m, 20H), 3.76-3.35 (m, 432H), 3.23 (s, 3H, OMe).

Example 5 PEG₂₀₀₀ polysulfate (PG506)

(A) A mixture of the imidate (15) (60 mg, 36.5 μmol) and PEG₂₀₀₀-OMe(110 mg, 55.0 μmol) was treated with TMSOTf as described for PEG₅₀₀₀-OMeto yield compound 17 as a colourless glass (96 mg, 74%). ¹H NMR (400MHz, CDCl₃) δ 5.28-5.13, 5.00-4.87, 4.27-3.40 (3m, many H,H1^(I-V),2^(I-V),3^(I-V),4^(I-V),5^(I-V),6a^(I-V),6b^(I-V),OCH₂CH₂O),3.34 (s, 3H, OMe), 2.15-1.94 (16s, 3H each, COMe).

(B) Compound 17 was deacetylated according to the general procedure toyield the PEG₂₀₀₀-OMe polyol as a colourless wax (63 mg, 81%). Thisresidue was used in the next reaction without further purification orcharacterisation.

(C) The product from (B) above was sulfonated according to the generalprocedure to yield the title compound (PG506) as a colourless powder (47mg, 68%). ¹H NMR (400 MHz, D₂O) δ 5.34-3.97 (m, 498H), 3.80-3.35 (m,81H), 3.23 (s, 3H, OMe).

Example 6 PG502

Azide 19

A solution of peracetate 12 (270 mg, 175 μmol), TMSN₃ (60 mg, 525 μmol)and SnCl₄ (200 μL of 1M in DCM) in anh. DCM (20 mL) was stirredovernight in the dark. Additional quantities (3 eq.) of TMSN₃ and SnCl₄were added and stirring was continued in the dark overnight again. Iceand NaHCO₃ (sat. aq.) were added and the mixture was extracted withEtOAc, washed with brine, evaporated and subjected to flashchromatography (10 g silica gel, gradient elution, 50:50 to 75:25EtOAc:Hx) to yield 218 mg (82%) of azide 19. ¹H NMR (400 MHz, CDCl₃) δ5.52 (d, 1H, J=2.0, H1^(I)), 5.29-5.12 (m, 8H), 5.02-4.87 (m, 7H),4.29-3.76 (m, 19H), 2.18-1.95 (m, 48H); ¹³C NMR (100 MHz, CDCl₃) δ170.5(9), 170.5(7), 170.5(6), 170.4, 170.3, 170.2, 170.1, 169.9(9),169.9(8), 169.9(5), 169.7(3), 169.6(9), 169.6(6), 169.6, 169.5, 169.3,99.3(0), 99.2(7), 99.1, 99.0, 88.1, 75.2, 75.1, 74.8, 71.1, 70.9, 70.8,70.6, 69.7, 69.5, 69.4, 69.2, 68.3, 67.3, 66.8, 66.7, 65.5(9), 65.5(8),62.6, 62.2, 62.0, 61.7, 20.8(8), 20.8(6), 20.8, 20.7, 20.6(2), 20.5(8),20.5(7), 20.5. HRMS calcd for C₆₂H₈₄N₃O₄₁ [M+H]⁺ 1526.4583, found1526.4557.

1-Deoxy-1-α-phenoxyacetamido peracetate 21

A solution of 19 (32 mg, 21 μmol), PPh₃ (11 mg, 42.6 μmol) andphenoxyacetyl chloride (7.3 mg, 43 μmol) in anh. acetonitrile (5 mL) wasstirred at 0° C. for 4 h then at r.t. overnight. EtOAc and NaHCO₃ (sat.aq.) were added and the organic layer was washed with brine then dried(MgSO₄) and subjected to flash chromatography (gradient elution 60:40 to90:10 EtOAc:Hx) to yield 11.4 mg (33%) of amide 21 with some remainingPPh₃/PPh₃O. ¹H NMR (400 MHz, CDCl₃) δ 7.36-7.32 (m, 2H), 7.18 (br d, 1H,J=8.1, NH), 7.00-6.90 (m, 3H), 5.79 (dd, 1H, J=3.8, 8.2, H1^(I)),5.32-4.97 (m, 15H), 4.60-3.76 (m, 21H), 2.20-1.95 (m, 48H, AcO). HRMScalcd for C₇₀H₉₂NO₄₃ [M+H]⁺ 1634.5045, found 1634.5002.

PG502

The peracetate 21 (11 mg, 6.7 μmol) was deacetylated and sulfonatedaccording to the general procedures to yield 6 mg (34% for 2 steps) ofPG502 after lyophilisation. ¹H NMR (400 MHz, D₂O, solvent suppressed) δ:7.30-7.21 (m, 2H, ArH^(m)), 6.96-6.84 (m, 3H, ArH^(o,p)), 5.56-3.59 (m,30H affected by suppression).

Example 7 PG503

1-Deoxy-1-α-biotinamidocaproamido peracetate 22

A mixture of 19 (70 mg, 46 μmol) and Adam's catalyst (2 mg) in 2:1EtOAc:EtOH (3 mL) was stirred under H₂ (100 psi) overnight, thenfiltered, evaporated and co-evaporated with anh. pyridine.Biotinamidocaproate N-hydroxysuccinimide ester (31 mg, 68 μmol) and 1 mLanh. pyridine were added and the mixture was heated to 60° C. for 3 dayswith stirring. The solution was evaporated and subjected to flashchromatography (9.4 g Et₃N washed silica gel, gradient elution 75:25EtOAc:Hx to 30:70 MeOH:EtOAc) to give 30.8 mg (36% over two steps) ofamide 22. ¹H NMR (400 MHz, CDCl₃) δ 7.41 (br d, 1H, J=9.4, NH), 6.47,6.17 (2×br s, 2×1H, imide NHs), 5.40 (br d, 1H, J=9.4, H1^(I)),5.40-4.90 (m, 16H), 4.52 (dd, 1H, J=4.9, 7.5, biotin-H4), 4.36-3.72 (m,20H), 3.25-3.12 (m, 3H), 2.91 (dd, 1H, J=5.0, 13.0, biotin-H5A), 2.75(d, 1H, J=12.9, biotin-H5B), 2.27-1.96 (m, 52H), 1.82-1.29 (m, 12H,alkyl chains).

PG503

The peracetate 22 (30 mg, 16.3 μmol) was deacetylated and sulfonatedaccording to the general procedures to yield 28 mg (61% for 2 steps) ofPG503 after lyophilisation. ¹H NMR (400 MHz, D₂O, solvent suppressed,affected by amide rotamers) δ 5.60-4.75 (m, 7H, sugar Hs), 4.68 (dd, 1H,J=4.7, 7.2, biotin-H4), 4.60-3.60 (m, 26H, sugar Hs), 4.21 (dd, 1H,J=4.4, 7.2, biotin-H3), 3.33-3.16 (m, 1H, biotin-H2), 3.07-2.97 (m, 3H,biotin-H5A+CH₂N), 2.92 (dd, 1H, J=4.9, 13.5, biotin-H5B), 2.33-2.14 (m,2H, COCH₂B), 2.09 (t, 2H, J=7.4, COCH₂A), 1.63-1.15 (m, 12H, alkylchains).

Example 8 PG505

Azide 31.

A solution of maltohexaose peracetate (500 mg, 273 μmol), TMSN₃ (83 mg,726 μmol) and SnCl₄ (145 μL of 1M in DCM) in anh. DCM (20 mL) wasstirred overnight in the dark. Addition quantities of TMSN₃ (50 μL) andSnCl₄ (100 μL of 1M in DCM) were added and stirring was continued in thedark overnight again. Ice and NaHCO₃ (sat. aq.) were added and themixture was extracted with EtOAc, washed with brine, evaporated andsubjected to flash chromatography (10 g silica gel, gradient elution,75:20 to 80:20 EtOAc:Hx) to yield 488 mg (98%) of azide 31. ¹H NMR (400MHz, CDCl₃) δ: 5.30-5.11 (m, 11H), 4.93 (t, 1H, J=9.9), 4.72 (dd, 1H,J=4.0, 10.5), 4.68-4.57 (m, 6H), 4.44-3.67 (m, 23H), 2.09-1.85 (m, 57H).¹³C NMR (100 MHz, CDCl₃) δ: 170.3(4), 170.3(1), 170.2(7), 170.2,170.1(4), 170.1(0), 170.0(7), 170.0, 169.6, 169.4, 169.3, 169.2(3),169.2(2), 169.1(7), 169.1(4), 169.1(1), 95.5(0), 95.4(5), 95.4, 95.3,87.1, 74.7, 73.9, 73.3, 73.2, 72.2, 71.4, 71.3, 71.2(4), 71.2(1), 70.2,70.1, 69.8, 69.0, 68.8, 68.7, 68.2, 67.7, 62.4, 62.3, 62.1(8), 62.1(6),62.0, 61.1, 30.0, 20.5(5), 20.5(3), 20.5(0), 20.4(6), 20.3(3), 20.2(8),20.2(4), 20.2(2).

PG505

The azide 31 (97 mg, 54 μmol) was deacetylated and sulfonated accordingto the general procedures to yield 66 mg (41% for 2 steps) of PG505after lyophilisation. ¹H NMR (400 MHz, D₂O, solvent suppressed) δ:3.69-5.78 (m, 42H affected by solvent suppression).

Example 9 PG515

6-Azido-6-deoxy-2,3,4-tri-O-benzoyl-α-D-mannopyranosyltrichloroacetimidate (34)

(A) H₂SO₄ (0.5 mL) was added to a cooled (0°) solution of the methylglycoside (32) [29] (1.52 g, 2.9 mmol) and Ac₂O (10 mL) in AcOH (5 mL)and the combined mixture stirred (0°→r.t., o/n). NaOAc (1.0 g) was addedportionwise until pH>5.0 and then the mixture was treated with MeOH (3mL). The mixture was filtered and the solvent evaporated andco-evaporated (toluene) prior to workup (EtOAc) and RSF (10-20%EtOAc/hexane) to yield presumably the acetate (33) as a colourless foam(1.12 g, 70%).

(B) Hydrazine acetate (196 mg, 2.13 mmol) was added to a stirredsolution of the acetate (33) (1.08 g, 1.94 mmol) in DMF (10 mL) and thecombined mixture heated (550, 15 min). The mixture was poured ontosaturated NaCl and extracted (EtOAc). The organic layer was evaporatedand subjected to RSF (10-30% EtOAc/hexane) to yield a colourless oil(888 mg). This residue was co-evaporated (2×100 mL CH₃CN) and used inthe next reaction without further purification or characterisation.

(C) DBU (3 drops) was added to a solution of the crude product from (B)(above) (888 mg) and Cl₃CN (2.0 mL, 20 mmol) in 1,2-DCE (8 mL) and thecombined mixture stirred (020 →r.t., 1 h). The mixture was filtered, thesolvent evaporated and the residue subjected to FC (10-30% EtOAc/hexane)to yield the imidate (34) as a colourless oil (777 mg, 61%, 2 steps). ¹HNMR (400 MHz, CDCl₃) δ 8.88 (br s, 1H, NH), 8.10-7.22 (m, 15H, ArH),6.56 (d, 1H, J_(1,2) 2.0 Hz, H1), 5.99 (dd, 1H, J_(3,4˜4,5) 9.6 Hz, H4),5.94-5.88 (m, 2H, H_(2,3)), 4.44 (ddd, 1H, J_(5,6) 2.8, 5.6 Hz, H5),3.54 (dd, 1H, J_(6,6) 13.6 Hz, H6), 3.47 (dd, 1H, H6). ¹³C NMR (100 MHz,CDCl₃) δ 165.61, 165.37, 159.95, 134.00, 133.92, 133.58, 130.25, 130.05,129.12, 129.04, 128.97, 128.91, 128.76, 128.74, 128.57, 94.62, 73.03,69.69, 68.90, 67.05, 51.06.

Benzyl(6-azido-6-deoxy-α-D-mannopyranosyl)-(1→3)-(α-D-mannopyranosyl)-(1→3)-(α-D-mannopyranosyl)-(1→2)-(α-D-mannopyranoside)(37)

(A) A mixture of the imidate (34) (93 mg, 141 μmol), the alcohol (35)(90 mg, 94.1 μmol) and mol. sieves (50 mg of 3 Å powder) in 1,2-DCE (3mL) was treated with TMSOTf (10 μL, 55.1 μmol) and the combined mixturestirred (0°→r.t, 20 min). Et₃N (100 μL) was introduced, the mixture wasfiltered and the solvent was evaporated. The residue subjected to FC(10-40% EtOAc/hexane) to yield the azide (36) as a colourless oil (68mg, 57%). ¹H NMR (400 MHz, CDCl₃) δ 8.80-7.12 (m, 65H, ArH), 6.01 (dd,1H, J_(3,4˜4,5) 9.9 Hz, H4^(III)), 5.96 (dd, 1H, J_(3,4˜4,5) 9.9 Hz,H4^(I)), 5.92 (dd, 1H, J_(3,4˜4,5) 9.6 Hz, H4^(II)), 5.83 (dd, 1H,J_(2,3) 3.3 Hz, H3^(I)), 5.79 (dd, 1H, J_(1,2) 2.0, J_(2,3) 3.3 Hz,H2^(II)), 5.70 (dd, 1H, J_(3,4˜4,5) 9.9 Hz, H4^(IV)), 5.50 (dd, 1H,J_(2,3) 3.3 Hz, H3^(IV)), 5.36 (d, 1H, J_(1,2) 1.7 Hz, H1^(III)), 5.29(dd, 1H, J_(2,3) 3.0 Hz, H2^(III)), 5.23 (d, 1H, H1^(II)), 5.18 (dd, 1H,J_(1,2) 1.9 Hz, H2^(IV)), 5.16 (d, 1H, J_(1,2) 1.6 Hz, H1^(I)), 4.87 (d,1H, H1^(IV)), 4.72-4.24 (m, 14H, H2^(I), H3^(II,III), H5^(I-III) ,H6^(I-III)), 3.99 (ddd, 1H, J_(5,6) 2.9, 3.4 Hz, H5^(IV)), 3.02 (dd, 1H,J_(6,6) 13.5 Hz, H6^(IV)), 2.83 (dd, 1H, H6^(IV)).

(B) The benzoate (36) (63 mg, 31 μmol) was transesterified according thegeneral procedure and chromatography (C18, 0-10% MeOH/H₂O) of theresidue to yield the tetrasaccharide (37) as a colourless glass (15 mg,62%). ¹H NMR (400 MHz, MeOD) δ 7.34-7.22 (m, 5H, ArH), 5.12 (d, 1H,J_(1,2) 1.5 Hz, H1a), 5.09 (d, 1H, J_(1,2) 1.7 Hz, H1b), 5.07 (d, 1H,J_(1,2) 1.6 Hz, H1c), 4.92 (d, 1H, J_(1,2) 1.9 Hz, H1d), 4.71, 4.48 (ABof AB quartet, J=11.7 Hz, CH₂Ph), 4.14 (dd, 1H, J_(2,3) 3.0 Hz, H2a),4.19 (dd, 1H, J_(2,3) 3.2 Hz, H2b), 3.96 (dd, 1H, J_(2,3) 3.4 Hz, H2c),3.94 (dd, 1H, J_(3,4) 9.4 Hz, H3b), 3.88-3.52 (m, 19H, H2d,H3a,c,d,H4a-d,H5a-d, H6a-d), 3.44 (dd, 1H, J_(5,6) 6.3, J_(6,6) 10.1 Hz,H6^(IV)).

PG515

The tetrasaccharide 37 (12 mg, 15.3 μmol) was sulfonated according tothe general procedures to yield 14 mg (38% for 2 steps) of PG515 afterlyophilisation. ¹H NMR (500 MHz, D₂O) δ 7.47-7.37 (m, 1H, ArH),5.45-4.02 (m, 29H, C1^(I-IV),2^(I-IV),3^(I-IV),4^(I-IV),5^(I-IV),6a^(I-IV),6b^(I-III), CH₂Ph),3.69-3.67 (m, 1H, H6^(IV)).

Example 10 PG509

Methyl3-O-(2,4,6-Tri-O-benzoyl-α-D-mannopyranosyl)-2,4,6-tri-O-benzoyl-α-D-mannopyranoside(39)

(A) A mixture of 3-O-allyl-2,4,6-tri-O-benzoyl-α-D-mannopyranosyltrichloroacetimidate [26] (410 mg, 0.57 mmol) and methyl2,4,6-tri-O-benzoyl-α-D-mannopyranoside [26] (300 mg, 0.51 mmol) in1,2-DCE (6 mL) in the presence of mol. sieves (700 mg of 3 Å powder) wastreated with TMSOTf (30 μL, 0.17 mmol) and the combined mixture stirred(0°→r.t, 30 min). Et₃N (100 μL) was introduced, the mixture was filteredand the solvent was evaporated. The residue subjected to FC (10-50%EtOAc/hexane) to yield, presumably, disaccharide 38 as a colourless oil.

(B) PdCl₂ (40 mg) was added to a solution of the product from (A) inMeOH (10 mL) and 1,2-DCE (10 mL) and the combined mixture was heated(70°, 40 min). The solvents were evaporated and the residue subjected toFC (10-50% EtOAc/hexanes) to yield the alcohol (39) as a colourless oil(316 mg, 68%, 2 steps). The ¹H and ¹³C NMR (CDCl₃) spectra were similarto those already reported in the literature [26].

Methyl(α-D-mannopyranosyl)-(1→3)-(α-D-mannopyranoside) (40)

The alcohol (39) (10 mg, 0.10 mmol) was transesterified according thegeneral procedure to yield the disaccharide (40) as a colourless oil (3mg, 85%), identical by NMR to that reported in the literature [30,31].

PG509.

The disaccharide 40 (25 mg, 70 μmol) was sulfonated according to thegeneral procedures to yield 27 mg (36%) of PG509 after lyophilisation.¹H NMR (400 MHz, D₂O) δ 5.26 (d, 1H, J_(1,2) 1.8 Hz; H1^(II)), 4.98 (dd,1H, J_(2,3) 2.4 Hz; H2^(II)), 4.87 (d, 1H, J_(1,2) 1.9 Hz; H1^(I)),4.60-4.55 (m, 1H; H3^(II)), 4.53 (dd, 1H, J_(2,3) 2.3 Hz; H2^(I)),4.41-4.19 (m, 5H; H4^(I), 4^(II), 6a^(I),6a^(II),6b^(II)), 4.15 (dd, 1H,J_(3,4) 9.3 Hz; H3^(I)), 4.06-3.91 (m, 3H; H5^(I), 5^(II), 6b^(I)), 3.29(s, 3H; OCH₃).

Example 11 PG508

Methyl3-O-[3-O-(2,4,6-Tri-O-benzoyl-α-D-mannopyranosyl)-2,4,6-tri-O-benzoyl-α-D-mannopyranosyl]-2,4,6-tri-O-benzoyl-α-D-mannopyranoside(42)

(A) A mixture of 3-O-allyl-2,4,6-tri-O-benzoyl-α-D-mannopyranosyltrichloroacetimidate (269 mg, 0.37 mmol) and the alcohol (39) (306 mg,0.31 mmol) in 1,2-DCE (5 mL) in the presence of mol. sieves (100 mg of 3Å powder) was treated with TMSOTf (20 μL, 0.11 mmol) and the combinedmixture stirred (0°→r.t, 30 min). Et₃N (100 μL) was introduced, themixture was filtered and the solvent was evaporated. The residuesubjected to FC (10-50% EtOAc/hexane) to yield, presumably, thetrisaccharide 41 as a colourless oil.

(B) PdCl₂ (40 mg) was added to a solution of the product from (A) inMeOH (10 mL) and 1,2-DCE (10 mL) and the combined mixture was heated(70°, 40 min). The solvents were evaporated and the residue subjected toFC (10-50% EtOAc/hexanes) to yield the alcohol (42) as a colourless oil(316 g, 70%, 2 steps). ¹H NMR (400 MHz, CDCl₃) δ 8.14-7.22 (m, 45H,ArH), 6.63 (dd, 1H, J_(1III,2III) 1.8, J_(2III,3III) , 3.3 Hz,H2^(III)), 5.94 (dd, 1H, J_(3III,4III) 10.0, J_(4III,5III) 10.0 Hz,H4^(III)), 5.84 (dd, 1H, J_(3II,4II) 9.9, J_(4II,5II) 9.9 Hz, H4^(II)),5.48 (dd, 1H, J_(3I,4I) 9.8, J_(4I,5I) 9.8 Hz, H4^(I)), 5.26 (d, 1H,J_(1I,2I) 1.9 Hz, H1^(I)), 5.22 (dd, 1H, J_(1II,2II) 2.1, J_(2II,3II)3.0 Hz, H2^(II)), 4.91 (d, 1H, H1^(III)), 4.90 (dd, 1H, J_(2I,3I) 3.2Hz, H2^(I)), 4.86 (dd, 1H, J_(1II,2II) 1.7 Hz, H1^(II)). 4.67-4.63 (,12H, H3^(I),3^(II),3^(III), 5^(I), 5^(II), 5^(III),6^(I),6^(II),6^(III)). ¹³C NMR (100 MHz, CDCl₃) δ 166.49, 166.38,166.25, 166.07, 165.94, 165.77, 165.63, 165.19, 165.15, 133.80, 133.60,133.61, 133.58, 133.52, 133.06, 130.22, 130.16, 130.09, 130.05, 130.16,129.97, 129.9, 129.88, 129.84, 129.51, 129.17, 129.01, 128.85, 128.63,128.53, 128.5, 128.46, 99.35, 99.24, 98.73, 76.48, 76.12, 72.45, 71.77,71.64, 69.93, 69.7, 69.01, 68.86, 68.6, 68.53, 67.82, 63.17, 62.79,62.41, 55.66; ESMS: m/z 1373.4 [M−Bz+H+ Na]⁺, 1269.4 [M−2Bz+2H+Na]⁺.

Methyl(α-D-mannopyranosyl)-(1→13)-(α-D-mannopyranosyl)-(1→13)-(α-D-mannopyranoside)(43)

The alcohol (42) (115 mg, 0.79 mmol) was transesterified according thegeneral procedure to yield the trisaccharide (43) as a colourless oil(35 mg, 86%), identical by NMR to that reported in the literature [32].HRMS: m/z 519.1862 [M+H]⁺, 541.1646 [M+Na]⁺.

PG508.

The trisaccharide 43 (25 mg, 49 μmol) was sulfonated according to thegeneral procedures to yield 36 mg (49%) of PG508 after lyophilisation.¹H NMR (400 MHz, D₂O) δ 5.26 (d, 1H, J_(1,2) 1.9 Hz; H1^(III)), 5.22 (d,1H, J_(1,2) 1.8 Hz; H1^(II)), 5.04 (dd, 1H, J_(2,3) 2.4 Hz; H2^(III)),4.89 (d, 1H, J_(1,2) 1.6 Hz; H1^(I)), 4.76-4.75 (m, 1H; H2^(II)),4.60-4.55 (m, 1H; H3^(III)), 4.55 (dd, 1H, J_(2,3) 3.1 Hz; H2^(I)), 4.50(dd, 1H, J_(3,4) 9.6, J_(4,5) 9.7 Hz; H4^(III)), 4.41-4.12, 4.04-3.91(m, 12H; H3^(II),4^(I),4^(II),5^(I-III),6a^(I-III),6b^(I-III)), 4.10(dd, 1H, J_(3,4) 9.5 Hz; H3^(I)), 3.29 (s, 3H; OCH₃).

Example 12 PG512

Benzyl(3-O-Allyl-α-D-mannopyranosyl)-(1→3)-(α-D-mannopyranosyl)-(1→3)-(α-D-mannopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-D-mannopyranoside) (44)

Sodium (small piece) was added to the nonabenzoate (28) (115 mg, 0.79mmol) in MeOH (6 mL) and the combined mixture stirred (r.t., o/n). Themixture was neutralised (Dowex 50×8, H⁺), filtered and the filtrateconcentrated and subjected to FC (0-10% MeOH/CH₂Cl₂) to yield thetetrabenzyl ether (44) as a colourless oil (89 mg, 64%). ¹H NMR (CD₃OD)δ 7.33-7.13 (m, 20H, ArH), 6.02-5.92 (m, 1H, CH═CH₂), 5.32-5.27,5.11-5.09 (2m, 2H, CH═CH₂), 5.10 (d, 1H, J_(1,2) 1.4 Hz, H1a), 5.09 (d,1H, J_(1,2) 1.5 Hz, H1b), 5.03 (d, 1H, J_(1,2) 1.2 Hz, H1c), 4.97 (d,1H, J_(1,2) 1.4 Hz, H1d), 4.74, 4.49 (2d, AB of ABq, J_(H,H) 10.9 Hz,PhCH₂-a), 4.67, 4.48 (2d, AB of ABq, J_(H,H) 11.8 Hz, PhCH₂-b), 4.65,4.58 (2d, AB of ABq, J_(H,H) 11.6 Hz, PhCH₂-c), 4.57, 4.51 (2d, AB ofABq, J_(H,H) 12.4 Hz, PhCH₂-d), 4.21-3.62 (m, 26H,H2^(I-IV),3^(I-IV),4^(I-IV),5^(I-IV), 6a^(I-IV), 6b^(I-IV), OCH₂CH═).

PG512

The tetrasaccharide 44 (23 mg, 21.5 μmol) was sulfonated according tothe general procedures to yield PG512 as a colourless powder (26 mg,61%). ¹H NMR (400 MHz, D₂O) δ 7.32-7.18, 7.00-6.98 (2m, 20H, ArH),5.88-5.78 (m, 1H, CH═CH₂), 5.30-5.23, 5.08-5.04, 4.91-4.90, 4.83-4.82,4.71-4.08, 4.00-3.89, 3.73-3.70, 3.62-3.45 (8m, 40H, CH═CH₂, OCH₂CH,H1-6^(I-IV), PhCH₂ ^(I-IV))

Example 13 PG513

A mixture of the tetrabenzyl ether (44) (62 mg, 50 μmol) and Pd(OH)₂ (10mg of 10% on C) in THF (1 mL) and H₂O (1 mL) was stirred under H₂ (100p.s.i.) (r.t., o/n). The mixture was filtered, concentrated andsubjected to FC (SiO₂; H₂O) to yield the propyl ether (45) as acolourless glass (32 mg, 73%). ¹H NMR (D₂O) δ 5.22 (br s, 1H, H1a), 5.00(d, 1H, J_(1,2) 1.7 Hz, H1b), 4.97 (d, 1H, J_(1,2) 1.6 Hz, H1c), 4.87(d, 1H, J_(1,2) 1.8 Hz, H1d), 4.11-4.07, 3.91-3.35 (2 m, 26H, H₂^(I-IV),3^(I-IV),4^(I-IV),5^(I-IV),6a^(I-IV),6b^(I-IV), OCH₂), 1.50-1.42(m, 2H, CH₂CH₃), 0.76 (t, 3H, J_(H,H) 7.2 Hz, CH₂CH₃).

PG513

The tetrasaccharide 45 (21 mg, 29.6 μmol) was sulfonated according tothe general procedures to yield PG513 as a colourless powder (29 mg,34%). ¹H NMR (D₂O) δ 5.61 (d, 1H, J_(1,2) 2.3 Hz; H1a), 5.61 (br s, 1H;H1b), 5.32 (d, 1H, J_(1,2) 1.8 Hz; H1c), 5.26 (d, 1H, J_(1,2) 2.0 Hz;H1d), 4.90-4.88, 4.77-4.31, 4.23-4.04, 3.98-3.81, 3.57-3.51, 3.41-3.36(6 m, 26H, OCH₂CH₂, H2-6^(I-IV)), 1.48-1.39 (m, 1H; CH₂CH₃), 0.76 (dd,1H, J_(H,H) 7.4 Hz; CH₂CH₃).

Example 14 PG510

The polyol 46 [31] (22 mg, 61.7 μmol) was sulfonated according to thegeneral procedure to yield PG510 as a colourless powder (46 mg, 70%).¹HNMR (D₂O) δ 5.10 (d, 1H, J_(1,2) 2.0 Hz; H1^(II)), 4.90 (d, 1H,J_(1,2) 2.0 Hz; H1^(I)), 4.78 (dd, 1H, J_(2,3) 3.0 Hz; H2^(II)), 4.73(dd, 1H, J_(2,3) 3.1 Hz; H2^(I)), 4.64-4.40 (m, 1H; H3^(II)), 4.52 (dd,1H, J_(3,4) 9.5 Hz; H3^(I)), 4.33-4.30 (m, 2H; H4^(II), 6a^(II)), 4.22(dd, 1H, J_(4,5) 9.7 Hz; H4^(I)), 4.12-4.04 (m, 2H; H5^(II), 6b^(II)),3.96-3.90 (m, 2H; H5^(I), 6a^(I)), 3.76 (dd, 1H, J_(5,6)b 8.6, J_(6a,6b)11.3 Hz; H6b^(I)), 3.31 (s, 3H; OCH₃).

Example 15 PG511

The polyol 47 [31] (20 mg, 56 μmol) was sulfonated according to thegeneral procedure to yield PG511 as a colourless powder (29 mg, 48%). ¹HNMR (D₂O) δ 5.36 (d, 1H, J_(1,2) 2.2 Hz; H^(II)), 4.90 (br s, 1H;H2^(II)), 4.87 (d, 1H, J_(1,2) 2.1 Hz; H1^(I)), 4.74 (dd, 1H, J_(2,3)3.0 Hz; H2^(II)), 4.58-4.40, 4.29-4.10, 3.88-3.85 (3 m, 10H,H3-6^(I,II)), 3.30 (s, 3H; OCH₃).

Example 16 PG514

Azide 18

(A) Boron trifluoride diethyl etherate (257 mg, 1.81 mmol) was slowlyadded to a solution of the peracetate 12 (700 mg, 0.453 mmol) and6-bromo-1-hexanol (492.7 mg, 2.721 mmol) in DCE (20 mL, 3A molecularsieves) and the mixture was stirred under argon at 60° C. for 72 h. Thesolution was cooled, neutralised with Et₃N, diluted with DCM (30 mL),washed with sat. NaHCO₃, dried (MgSO₄) and subjected to flashchromatography (silica, gradient elution, 40:60 to 100:0 EtOAc:Hx) toafford 340 mg (0.204 mmol, 45.0%) of the 6-bromohexyl glycoside. ¹H NMR(400 MHz, CDCl₃) δ: 5.25-5.08 (m, 8H), 4.98-4.81 (m, 8H), 4.25-3.70 (m,19H), 3.607 (dt, 1H, J=9.553, J=6.635, OCH₂A), 3.354 (dt, 1H, J=9.641,J=6.637, OCH₂B), 3.33 (t, 2H, J=6.700, CH₂Br), 2.104, 2.096, 2.09, 2.06,2.043, 2.038, 2.036, 2.033, 2.029, 2.02, 2.01, 1.97, 1.95, 1.94 and 1.90(16×S, 48H, OAc), 1.85-1.74 (m, 2H, CH₂), 1.59-1.46 (m, 2H, CH₂),1.44-1.35 (m, 2H, CH₂), 1.35-1.25 (m, 2H, CH₂); ¹³C NMR (CDCl₃, 100MHz): 170.42, 170.41, 170.39, 170.28, 170.16, 170.07, 169.96, 169.94,169.83, 169.77, 169.58, 169.52, 169.45, 169.36, 169.25 (19×CO), 99.10,98.83, 98.75, 98.01 (sugar-C1), 76.96, 75.00, 74.83, 74.75, 70.96,70.82, 70.70, 70.08, 69.49, 69.28, 69.16, 68.24, 68.17, 68.04, 67.20,66.65, 66.60, 66.09, 65.44, 62.41, 62.31, 61.86, and 61.54 (sugarcarbons excluding sugar-C1 and bromohexyl-CH₂O), 33.49, 32.32, 29.43,28.92, 27.59, 25.12 (6×bromohexyl-CH₂), 20.73, 20.71, 20.68, 20.62,20.56, 20.47, 20.44, 20.41, (Ac-CH₃), 13.85 (CH₂Br).

(B) A solution of 6-bromohexyl glycoside from (A) (340 mg, 0.204 mmol)and sodium azide (66 mg, 1.02 mmol) in DMF (4 mL) was heated at 100° C.for 48 h. TLC analysis of the crude mixture indicated no change.Tetrabutylammonium idiode (20 mg) was then added and the mixture allowedto react for a further 48 h. The crude mixture was cooled and subjectedto flash chromatography (0:100 to 5:95 DCM:MeOH) to afford 21.1 mg(0.013 mmol, 6.4%) of azide 18.

PG514

(A) The azide 18 (21.1 mg, 0.013 mmol) was deacetylated under standardZemplén conditions (2 mL MeOH) to afford 12.6 mg (0.013 mmol, 102%) ofpolyol 48.

(B) The polyol 48 (12.6 mg, 13.2 μmol) was treated withSO₃.trimethylamine according to the general sulfation procedure to yieldPG514 as a colourless powder (18.4 mg, 54%). ¹H NMR (D₂O, 400 MHz):5.40-4.69 (m, 8H), 4.68-3.41 (m, 27H), 3.22 (t, 2H, J=6.5), 1.51 (br s,5H), 1.29 (br s, 5H).

Biological Testing of Compounds

Growth Factor Binding Assays

Binding affinities of ligands for the growth factors FGF-1, FGF-2 andVEGF were measured using a surface plasmon resonance (SPR) basedsolution affinity assay. The principle of the assay is that heparinimmobilised on a sensorchip surface distinguishes between free and boundgrowth factor in an equilibrated solution of the growth factor and aligand. Upon injection of the solution, the free growth factor binds tothe immobilised heparin, is detected as an increase in the SPR responseand its concentration thus determined. A decrease in the free growthfactor concentration as a function of the ligand concentration allowsfor the calculation of the dissociation constant, K_(d). It is importantto note that ligand binding to the growth factors can only be detectedwhen the interaction involves the HS binding site, thus eliminating thechance of evaluating non-specific binding to other sites on the protein.A 1:1 stoichiometry has been assumed for all protein:ligandinteractions.

For the testing of growth factor binding activity, heparin-coatedsensorchips were used. Their preparation, via immobilisation ofbiotinylated BSA-heparin on a streptavidin-coated sensorchip, has beendescribed. [5] Heparin has also been immobilised via aldehyde couplingusing either adipic acid dihydrazide or 1,4-diaminobutane. For eachK_(d) measurement, solutions were prepared containing a fixedconcentration of protein and varying concentrations of the ligand inbuffer. Ligands binding to FGF-1 and VEGF were measured in HBS-EP buffer(10 mM HEPES, pH 7.4, 150 mM NaCl, 3.0 mM EDTA and 0.005% (v/v)polysorbate 20), while binding to FGF-2 was measured in HBS-EP buffercontaining 0.3 M NaCl. [5] Prior to injection, samples were maintainedat 4° C. to maximise protein stability. For each assay mixture, 50-200μL of solution was injected at 5-40 μL/min and the relative bindingresponse measured. All surface binding experiments were performed at 25°C. The surface was regenerated by injection of 40 μL of 4M NaCl at 40μL/min, followed by injection of 40 μL of buffer at 40 μL/min.

Sensorgram data were analysed using the BIAevaluation software(BIAcore). Background sensorgrams were subtracted from experimentalsensorgrams to produce curves of specific binding, and baselines weresubsequently adjusted to zero for all curves. Standard curves relatingthe relative response value to the injected protein concentration arelinear, indicating that the binding response is proportional to theprotein concentration, and thus suggesting that the binding experimentswere conducted under mass transport conditions. [34] Therefore, therelative binding response for each injection can be converted to freeprotein concentration using the equation.$\lbrack P\rbrack = {\frac{r}{r_{m}}\lbrack P\rbrack}_{total}$where r is the relative binding response and r_(m) is the maximalbinding response.

Binding equilibria established in solution prior to injection wereassumed to be of 1:1 stoichiometry. Therefore, for the equilibrium,P+L

P·Lwhere P corresponds to the growth factor protein, L is the ligand, andP·L is the protein:ligand complex, the equilibrium equation is$K_{d} = \frac{\lbrack P\rbrack\lbrack L\rbrack}{\left\lbrack {P \cdot L} \right\rbrack}$and the binding equation [5] can be expressed as$\lbrack P\rbrack = {\lbrack P\rbrack_{total} - \frac{\left( {K_{d} + \lbrack L\rbrack_{total} + \lbrack P\rbrack_{total}} \right)}{2} + \sqrt{\frac{\left( {K_{d} + \lbrack L\rbrack_{total} + \lbrack P\rbrack_{total}} \right)^{2}}{4} - {\lbrack L\rbrack_{total}\lbrack P\rbrack}_{total}}}$

The K_(d) values given are the values fit, using the binding equation,to a plot of [P] versus [L]_(total). Where K_(d) values were measured induplicate, the values represent the average of the duplicatemeasurements. It has been shown that GAG mimetics that bind tightly tothese growth factors, e.g., PI-88, elicit a biological response invivo.[5]

Heparanase Inhibition Assays

The heparanase assays were performed using a Microcon ultrafiltrationassay. The assays rely on the principle of physically separating heparansulfate (HS) that has been digested by heparanase from native HS todetermine heparanase activity. The assay uses ultrafiltration devices(Microcon YM-10) to separate the smaller heparanase-cleaved HS fragmentsfrom native HS.

A reaction was set up with a volume of 90 μL,

-   -   40 mM acetate buffer (pH 5.0)    -   0.1 mg/mL BSA    -   90 ng heparanase    -   2.5 μM ³H labelled HS    -   various concentrations of inhibitors.

The reactions were set up with all components except the ³H labelled HSand allowed to equilibrate for 10 min at 22° C. The assays were theninitiated by adding the HS and immediately 20 μL was taken, mixed with80 μL of 10 mM phosphate (pH 7.0) and the 100 μL transferred to aMicrocon YM-10 concentrator which was then centrifuged at approximately14000 g for 5 min. The solution that passed through the membrane(filtrate) was retained. This sample was considered the time=0 sample.The assays (now 70 μL in volume) were allowed to react at 22° C. for 2.5h and then the filtration step was repeated for three aliquots of 20 μLfrom each assay.

The time=0 filtrate and the three 2.5 h filtrate samples were countedfor ³H. The difference between the time=0 and the averaged 2.5 h samplesgave the amount of heparanase activity. All inhibition assays were runwith a heparanase standard assay which was identical to the assaycomposition above except no inhibitor was present and the amount ofheparanase inhibition in the other assays determined by comparison withthis standard. The IC₅₀ for PI-88 in this assay is 0.98 μM.

Antiviral Assays

Monolayer cultures of African green monkey kidney cells[35] and herpessimplex virus (HSV-1) KOS321 strain[36] were used throughout. Theantiviral assays on the compounds were performed as described by Nyberget al.[13] Briefly, the effects of the compounds on the infection ofcells by exogenously added virus were tested by mixing serial fivefolddilutions of compound (at 0.032-20 μM) with approximately 200 plaqueforming units of the virus. Following incubation of the virus andcompound for 10 min at room temperature, the mixture was added to thecells and left on the cell monolayer for 2 h at 37° C. Subsequently, theinoculum was aspirated and replaced with an overlay medium of 1%methylcellulose solution in Eagle's minimum essential medium (EMEM). Theviral plaques that developed after incubation of cells for 3 days at 37°C. were stained with 1% crystal violet solution and counted. The effectsof the compounds on cell-to-cell spread of HSV-1 were tested by addingserial fivefold dilutions of compound (at 0.032-20 μM) in the serum-freeoverlay medium to cells after their infection with HSV-1. Afterincubation of the compound with the cells for 3 days at 37° C., theimages of 20 plaques were captured and subjected to area determinationusing IM500 software (Leica). The results on viral infection of cellsand on viral cell-to-cell spread are shown in FIGS. 1A and 1B,respectively, whilst the derived IC₅₀ values are presented in Table 1.

Results

The results of the tests as described in the preceding section arepresented in Table 1. TABLE 1 Heparanase HSV-1 HSV-1 cell-to- K_(d) aFGFK_(d) bFGF K_(d) VEGF Inhibition Infectivity cell spread Compound (pM)(nM) (nM) (IC₅₀, μM) (IC₅₀, μM) (IC₅₀, μM) PG500 120 ± 25 86 ± 7 1.72 ±0.19 1.83 ± 0.483 2 1 PG501 144 ± 8  68.3 ± 2.9 1.67 ± 0.11 1.64 ± 0.4061 0.4 PG502 660 ± 40 112 ± 9  7.1 ± 0.6 2.02 ± 0.284 7 5 PG503 390 ± 7084 ± 8 7.2 ± 0.6 1.85 ± 0.311 2 3 PG504 361 ± 28 150 ± 9  8.1 ± 0.6 6.03± 1.05  Not 11 tested PG505 1960 ± 300 137 ± 12 4.8 ± 0.4 1.04 ± 0.147 36 PG506  88 ± 17 114 ± 13 3.5 ± 0.8 2.12 ± 0.152 10 7Pharmacokinetic EvaluationPreparation of [³⁵S]-labelled Compounds

The polyol precursors for PG500, 501, 503, 504, 506 and PI-88 (2 mg ofeach) were desiccated under vacuum over P₂O₅ for 3 days. Into each vialwas syringed 50 μL of a stock solution of 1.77 mg (2.0 mCi) of³⁵SO₃.pyridine complex and 2 mg SO₃.Me₃N in 300 μL of anhydrous DMF(Aldrich, redried over freshly ignited 3A molecular sieves). A further600 μL of anhydrous DMF was added to the SO₃ vial and was distributed toeach sample vial. The samples were heated to 60° for 66 hr. SO₃.Me₃N (14mg in 300 μL anhydrous DMF) was added to each vessel and the resultingsolutions were heated to 60° overnight. The vials were cooled to roomtemperature and stored at −80° C. awaiting purification.

Each sample was quenched by addition of Na₂CO₃ (sat. aqu. adjusted to pH8-9), evaporated to dryness and subjected to SEC (Biogel P2, 2.6×90 cm,flow rate 30 mL/hr, 5 min/fraction). Fractions containing desiredmaterial were detected using a G-M counter and DMB test followed by CE.The results are summarized in Table 2. TABLE 2 Summary of Results forRadio-labelling experiments Quantity Radio-chemical Specific activityCompound isolated (mg) purity (μCi/mg) PI-88 2.8 99.0% 38.01 PG500 2.198.7% 29.19 PG501 1.7 98.0% 6.56 PG503 1.0 99.2% 5.49 PG504 5.0 98.3%6.47 PG506 3.6 99.0% 26.23Pharmacokinetic Studies

Male Sprague Dawley rats (250-350 g) were used. The animals were allowedfree access to food and water before and during the experiments, duringwhich they were maintained unrestrained in metabolism cages. Rats wereanaesthetized with isoflurane (Forthane®). A catheter was inserted inthe external jugular vein via an incision in the neck, and was passedunder the skin to a second incision in the skin of the back (midlinevicinity of the scapulae). This was then exteriorised with theprotection of a light metal spring. The incision was closed and thespring fixed to the skin with Michel sutures so that the rats had fullrange of movement. The animals were carefully monitored during recovery(1-4 h).

Stock dosing solutions were prepared by mixing appropriate amounts ofunlabelled and radiolabelled drug (dissolved in phosphate-bufferedsaline) to give a total drug concentration of 1.25 mg/mL. All doses wereadministered as a bolus intravenous injection of 2.5 mg/kg in a dosevolume of 2 mL/kg. The total amount of radioactivity administered toeach rat was 0.5-10 μCi. The dose level used in this study is 10-foldlower than the no-effect dose previously established for acute toxicityof PI-88. Blood samples (˜250 μL) were collected predose and at 5, 15,30, 45 min, and 1, 1.5, 2, 4, 8, 12, 24, 36 and 48 h after dosing. Theblood samples were immediately centrifuged and the plasma collected. Atcompletion of the experiments, the animals were killed by a lethaloverdose of IV pentobarbitone anaesthetic (Nembutal®). Urine wascollected from each animal at intervals of 0-12 h, 12-24 h and 24-48 hafter dosing. Cage washings (˜15 mL of deionised water) were alsocollected. At the end of the experiment, bladder contents were aspiratedfrom each animal and added to the 24-48 h voidings. Faeces werecollected over the same time intervals as the urine.

Aliquots of plasma (100 μL), urine and cage washings (500 μL) weretransferred directly to 6 mL polypropylene scintillation vials fordetermination of radioactivity. Faeces collected during each time period(from one animal dosed with each compound) were weighed and homogenisedin 4 volumes of deionised water using a mechanical homogeniser.Approximately 1 g (accurately weighed) of this slurry was transferred toa 20 mL glass scintillation vial, 2 mL of tissue solubiliser added andthe vials capped and incubated at 60° C. for at least 24 h. Radioactvitywas measured following mixing of samples with Packard Ultima Gold liquidscintillation counting cocktail (2.0 mL for plasma and dose, 5.0 mL forurine and cage washings, 10 mL for faeces). Counting was conducted on aPackard Tr-Carb liquid scintillation counter. Any result less than threetimes the background was considered less than the lower limit ofquantitation not used in calculations. Plasma, urine and cage washingswere counted in triplicate within 5 days of collection and were notcorrected for radiochemical decay. Faeces were processed as a batch atthe completion of the study and the counts from these samples werecorrected for radiochemical decay. Plasma pharmacokinetic parameterswere calculated using PK Solutions 2.0 software (Summit ResearchServices, Ohio) and are presented in Table 3. TABLE 3 Pharmacokineticparameters determined for ³⁵S-labelled compounds following ivadministration to male Sprague Dawley rats PI-88 PG500 PG501 PG503 PG504PG506 n 4 4 4 4 4 4 C₀ 17.7 ± 2.23 20.5 ± 1.3 35.6 ± 3.1 14.0 ± 0.8430.5 ± 2.3 17.1 ± 1.8 (μg-eq/mL) AUC_(0-12 h) 9.6 ± 1.9 12.6 ± 1.2 29.7± 3.4  6.5 ± 0.4^(b) 14.7 ± 1.2  6.2 ± 1.0^(a) (μg-eq/h · mL) t_(1/2)*(h)^(c) 0.83 ± 0.09  0.83 ± 0.02  1.10 ± 0.09 0.79 ± 0.03  2.81 ± 0.04 0.59 ± 0.01 k* (h⁻¹) 0.844 ± 0.096  0.836 ± 0.024  0.633 ± 0.053 0.879± 0.028  0.247 ± 0.003  1.17 ± 0.024 Cl* (mL/h/kg)  250 ± 27.6  199 ±13.2 83.6 ± 9.1  380 ± 24.3^(b)   172 ± 11.8   404 ± 59.5 Vd* (mL) 43.1± 1.9  38.4 ± 3.8 22.9 ± 2.2 55.1 ± 2.6  24.9 ± 2.9 44.5 ± 4.5 UrinaryRecovery 59.1 ± 13.1 39.3 ± 5.5 41.8 ± 1.5 80.5 ± 3.9  66.5 ± 9.4 79.1 ±3.6 (% dose)*Apparent values.^(a)Calculated over 0-8 h post dose interval only.^(b)Calculated over 0-4 h post dose interval only.^(c)Calculated over the 0.75-4.0 h post dose interval for PI-88, PG500,PG501, PG503 and PG506; calculated over the 4.0-12 h post dose intervalfor PG504.

The results presented in Table 1 demonstrate that the broad range ofcompounds embraced by the invention possess heparanase inhibitoryactivity and have strong affinity for GAG-binding growth factors and canthus serve as modulators of the activity of such factors in a similarmanner to PI-88. In addition, the compounds have similar antiviralactivity to PI-88. The results presented in Table 3 illustrate that thecompounds have altered pharmacokinetic properties compared to PI-88.

The foregoing embodiments are illustrative only of the principles of theinvention, and various modifications and changes will readily occur tothose skilled in the art. The invention is capable of being practicedand carried out in various ways and in other embodiments. It is also tobe understood that the terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

The term “comprise” and variants of the term such as “comprises” or“comprising” are used herein to denote the inclusion of a stated integeror stated integers but not to exclude any other integer or any otherintegers, unless in the context or usage an exclusive interpretation ofthe term is required.

Any reference to publications cited in this specification is not anadmission that the disclosures constitute common general knowledge inAustralia.

REFERENCES

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1. A compound of the general formula:X—[Y]_(n)-Z-UR¹ wherein: X, Y and Z are each a monosaccharide unit witha group UR bonded via a single or multiple bond to each non-linkingcarbon of X, Y and Z, except carbon-1 of monosaccharide Z which bearsUR¹ bonded via a single or multiple bond; n is an integer having a valueof 0-6; each U is independently C, N, S or O or their higher oxidationstates, including CO, COO, NO, NO₂, S(O), S(O)O; each R is independentlySO₃M or H, where M is any pharmaceutically acceptable cation or is anyalkyl, aryl, acyl, aroyl, alkyl sulfonyl, aryl sulfonyl, PEG, a PEGderivative, H or the group

where independently in each AB group, A is O or NH, and B is H, or Mwhere M is as defined above, or al alkyl, aryl or any other suitablegroup; R¹ is SO₃M, H, alkyl, aryl, acyl, aroyl, alkyl sulfonyl, arylsulfonyl, PEG or a PEG derivative, or R¹ together with U is N₃ or asubstituted triazole or derivative, or a substituted tetrazole orderivative, or a substituted aryl or derivative, or a substitutedheteroaryl or derivative; with the provisos that: at least one of UR¹ orUR is not OSO₃M, NSO₃M, OH or OPO₃M₂, and at least 50% of the R groupsare SO₃M.
 2. A compound of the general formula:

wherein: n is an integer having a value of 0-6; U is C, No S or O ortheir higher oxidation states, including CO, COO, NO, NO₂, S(O), S(O)O;each R is independently SO₃M or H4, where M is any pharmaceuticallyacceptable cation or is any alkyl, aryl, acyl, aroyl, alkyl sulfonyl,aryl sulfonyl, PEG, a PEG derivative, H or the group

where independently in each AB group, A is O or NH, and B is H, or Mwhere M is as defined above, or an alkyl aryl or any other suitablegroup; R¹ is SO₃M, H, alkyl, aryl, acyl, aroyl, alkyl sulfonyl, arylsulfonyl, PEG or a PEG derivative, or R¹ together with U is N₃ or asubstituted triazole or derivative, or a substituted tetrazole orderivative, or a substituted aryl or derivative, or a substitutedheteroaryl or derivative; with the provisos that: when U is O or N, atleast one of R¹ or K is not SO₃M, H or PO₃M₂, and at least 50% of the Rgroups are SO₃M.
 3. The compound of claim 1 or claim 2, wherein M issodium.
 4. The compound of claim 1 or claim 2, wherein n is
 3. 5. Thecompound of claim 1 or claim 2, wherein R¹ is n-octyl.
 6. The compoundof claim 1 or claim 2, wherein 70 to 100% of the R groups comprise SO₃M.7. A compound according to claim 1, wherein said compound is PG500,PG501, PG502, PG503, PG504, PG506, PG507, PG508, PG509, PG510, PG511,PG512, PG513, PG514 as hereinbefore described.
 8. A pharmaceutical orveterinary composition for the prevention or treatment in a mammaliansubject of a disorder resulting from angiogenesis, metastasis,inflammation, coagulation/thrombosis, raised blood triglyceride levels,microbial infection and/or cardiovascular disease, which compositioncomprises at least one compound according to claim 1 or claim 2 togetherwith a pharmaceutically or veterinarially acceptable carrier or diluentfor said at least one compound.
 9. The composition according to claim 9which further includes a pharmaceutically or veterinarially acceptableexcipient, buffer, stabiliser, isotonicising agent, preservative orantioxidant.
 10. The composition according to claim 9, wherein saidcompound is present therein as an ester, a free acid or base, a hydrate,or a prodrug.
 11. Use of a compound according to claim 1 or claim 2 inthe manufacture of a medicament for the prevention or treatment in amammalian subject of a disorder resulting from angiogenesis, metastasis,inflammation, coagulation/thrombosis, raised blood triglyceride levels,microbial infection and/or cardiovascular disease.
 12. The use accordingto claim 12, wherein said mammalian subject is a human subject.
 13. Amethod for the prevention or treatment in a mammalian subject of adisorder resulting from angiogenesis, metastasis, inflammation,coagulation/thrombosis, raised blood triglyceride levels, microbialinfection and/or cardiovascular disease, which method comprisesadministering to the subject an effective amount of at least onecompound according to claim 1 or claim 2, or a composition comprisingsaid at least one compound.
 14. The method according to claim 14 whereinsaid mammalian subject is a human subject.
 15. The method according toclaim 14, wherein said disorder resulting from angiogenesis is aproliferative retinopathy or angiogenesis resulting from the growth of asolid tumour.
 16. The method according to claim 14, wherein saiddisorder resulting from inflammation is rheumatoid arthritis, multiplesclerosis, inflammatory bowel disease, allograft rejection or chronicasthma.
 17. The method according to claim 14, wherein said disorderresulting from coagulation and/or thrombosis is deep venous thrombosis,pulmonary embolism, thrombotic stroke, peripheral arterial thrombosis,unstable angina or myocardial infarction.